EP3015542A1 - Modular system and method for continuous, germ reduced production and/or processing of a product - Google Patents
Modular system and method for continuous, germ reduced production and/or processing of a product Download PDFInfo
- Publication number
- EP3015542A1 EP3015542A1 EP15166686.4A EP15166686A EP3015542A1 EP 3015542 A1 EP3015542 A1 EP 3015542A1 EP 15166686 A EP15166686 A EP 15166686A EP 3015542 A1 EP3015542 A1 EP 3015542A1
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- EP
- European Patent Office
- Prior art keywords
- product
- germ
- filter
- steps
- protein
- Prior art date
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Classifications
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-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M37/00—Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/12—Purification
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/16—Sterilization
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/12—Addition of chemical agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2626—Absorption or adsorption
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/26—Further operations combined with membrane separation processes
- B01D2311/2649—Filtration
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2315/00—Details relating to the membrane module operation
- B01D2315/16—Diafiltration
Definitions
- the invention relates to a modular system and a method for the continuous, germ-reduced production and / or preparation of a product from a heterogeneous cell culture fluid mixture.
- proteins are purified in the biotechnological production in batch (English batch : “stack"). This means that the individual production cycles are handled in batches, discontinuously, and after the completion of a production cycle, the product is removed from the whole. For a new production then a newer product cycle or batch must be started separately.
- the loss of use of the reactors due to the provisioning procedures may be on the order of the reactor availability, in particular in the case of short periods of use and frequent product changes.
- WO2012 / 078677 describes a method and a plant for the continuous preparation of biopharmaceutical products by chromatography and their integration in a production plant, especially in a disposable plant. Even though WO2012 / 078677 Provides approaches for the continuous production of biopharmaceutical and biological products, the disclosed solution is not sufficient in practice. WO2012 / 078677 also does not disclose the use of a sterilized chromatography column.
- US 2014/0255994 A1 discloses an integrated continuous process for the production of therapeutic proteins. US 2014/0255994 A1 does not disclose the feature that sterilized chromatography columns could be used in such a process.
- EP 2 182 990 A1 discloses a method of sterilizing chromatography columns by using hot steam.
- a continuous process in the context of the invention means any process for carrying out at least two process steps in series, in which the output stream of an upstream step is conveyed into a downstream step.
- the downstream step begins processing the product stream before the upstream step is completed.
- a part of the product stream is always carried in the production plant and becomes referred to as "continuous product stream”.
- a continuous transport or transfer of a product stream from an upstream unit to a downstream unit means that the downstream unit is already operating before the upstream unit is taken out of service, ie, two successive units simultaneously process the product stream that flows through it.
- germ-reduced in the context of the invention means a state of reduced germ count, ie a number of microorganisms per unit area or volume unit of almost zero, which can be achieved by a suitable germ reduction method, this germ reduction method can be selected from gamma irradiation, beta Irradiation, autoclaving, ethylene oxide (ETO) treatment and steam-in-place (SIP) treatment.
- the term "disposable article” means that the product-contacted parts in question, in particular apparatuses, containers, filters and connecting elements, are disposable with disposable use, these containers being made of plastic as well as plastic also be made of metal.
- the term also includes reusable articles, for example of steel, which are used only once in the process according to the invention and are then no longer used in the process. These reusable articles, e.g. made of steel, are then referred to in the context of the invention "used as disposable” items. Such used disposable articles can then also be referred to as “disposable” or “single-use” articles in the process according to the invention ("SU technology").
- SU technology single-use
- product stream in the context of the invention means the particle-free fluid from a heterogeneous cell culture fluid mixture which contains the product, as well as the result of every other process steps of the process according to the invention, ie the product stream after filtration, after chromatography, after virus depletion after ultrafiltration, after diafiltration, or after further steps of the process according to the invention, these product streams then being able to have different concentrations and degrees of purity.
- virus depletion in the context of the invention means a reduction in the concentration of active viruses per unit volume of the fluid to be treated, to the complete inactivation and / or removal of the viruses contained in the fluid to be treated.
- microbicide in the context of the invention means a substance which can slow or completely inhibit the growth of microorganisms, it being possible to use this microbicide in the form of a microbicidal buffer, in particular within the scope of the process according to the invention during ultrafiltration.
- bubble trap in the context of the invention means a device for collecting gas bubbles, wherein the relevant fluid is degassed.
- module means within the scope of the invention that the individual steps of the method according to the invention can be carried out in separate, interconnected modules, wherein the modules are preconfigured and germ-reduced and can be connected together in different combinations.
- module in the context of the invention means a series of interconnected modules ("units") for carrying out at least two downstream and / or upstream steps in which a fluid ("product stream") can be conveyed.
- the units are suitable for the continuous performance of one step and can be operated with a continuous fluid flow ("product flow”).
- product flow a continuous fluid flow
- the individual modules of the "modular system” can be interconnected in any combination. Examples of modules within the meaning of the invention are the filtration module 2, the chromatography module 3, the ultrafiltration module 6, the diafiltration module 7 and the dialysis module 8.
- closed in the context of the invention means the mode of operation of the method according to the invention and the modular system according to the invention, which are operated such that the product which is produced and / or processed with this method and this modular system is not exposed to the room environment becomes.
- materials, objects, buffers and the like can be added externally, but this addition takes place in such a way that exposure of the produced and / or processed product to the room environment is avoided ,
- a biological product such as a protein, for example, a therapeutic protein.
- the nutrient solution is also an ideal growth medium for microorganisms, such as bacteria and spores, resulting in a problem with regard to the unwanted growth of such microorganisms.
- This undesirable growth of microorganisms becomes a problem, especially with longer maturities, because the nutrient solution becomes more and more contaminated with increasing runtime of the process, up to an exponential growth of microorganisms and thus total loss of the respective batch of biological product produced.
- the particle-free fluid from a heterogeneous cell culture-fluid mixture provided in step a) can preferably originate from a continuous perfusion and fermentation process, for example a cell culture or a perfusion reactor. In this case, even more than one perfusion reactor can be operated in parallel, for example two perfusion reactors.
- the fluid may be initially removed by suitable cell retention systems, such as a slant plate separator (settler), through which a majority of the cells can be retained. Subsequent filtration and / or centrifugation steps or other suitable separation processes may then free the fluid from particles contained in the fluid, thereby yielding a particle-free fluid in which the biopharmaceutical biological macromolecular product resides.
- suitable cell retention systems such as a slant plate separator (settler)
- filtration and / or centrifugation steps or other suitable separation processes may then free the fluid from particles contained in the fluid, thereby yielding a particle-free fluid in which the biopharmaceutical biological macromolecular product resides.
- the filtration step (b) may be, for example, a filtration of the particle-free fluid obtained after step (a), whereby a filtrate is obtained.
- the process according to the invention can also comprise further filtration steps at suitable points of the process.
- the filtration step b) can be carried out by suitable filtering methods, eg a 0.2 ⁇ m filter or several filters operated in parallel.
- a suitable filter for the filtration step is for example a Sartoguard NF 0.2 ⁇ m filter, several of which can be operated in parallel.
- the filtration step (b) comprises a depth filter with an additional possibility of depleting contaminants such as DNA, ProtA, HCP. These may be depth filters with sufficient zeta potential (Zetapor 3M, Posidyne, Pall) or activated carbon activated carbon filters (Millistak Merck Millipore).
- the at least two chromatographic steps for purifying the product of step c) comprise purification via at least two chromatography columns and / or membrane adsorbers.
- the chromatographic columns and / or membrane adsorbers may have any suitable binding principle, such as affinity of the product for a ligand, ionic interactions, metal chelate bonding, hydrophobic interactions or van der Waals forces.
- the first chromatographic step of the at least two chromatography steps may be affinity chromatography (eg, a ligand having affinity for the product, such as protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, IgG different recombinant protein which has an affinity for the product).
- affinity chromatography eg, a ligand having affinity for the product, such as protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, IgG different recombinant protein which
- the process according to the invention is flexible here and can comprise in step c), depending on the degree of purity and concentration of the product to be achieved, any suitable chromatography principle in any order.
- the technical effect of using at least two chromatographic steps over at least two chromatography columns and / or membrane adsorbers according to step c), is that usually a single chromatography step does not sufficiently clean the contaminants such as host cell impurities, aggregates, DNA, protein A etc. can ensure.
- this allows continuous production in a chromatographic step, since at least one chromatography column and / or membrane adsorber can be loaded with unpurified product while at least one other chromatography column and / or membrane adsorber can be regenerated or eluted, thereby providing a continuous and effective procedure of the method can be achieved.
- one or more further steps for adjusting the pH and / or for adjusting the conductivity and / or filtration take place between the at least two chromatographic steps in (c) and / or after the virus inactivation in step (d) - and / or concentration steps and / or a buffer exchange. This allows a process-customizable driving style of the process.
- the at least one virus removal of step d) can be carried out in particular by adjusting the pH of the particle-free fluid, preferably to a pH of ⁇ 4.0.
- Setting the pH of the particle-free fluid to be inactivated to ⁇ 4.0 can be carried out, for example, by adding HCl solution. The addition is typically in advance of the virus depletion device.
- the pH is adjusted to> 4 with a base, for example, sodium hydroxide solution (NaOH) to terminate the virus depletion.
- the at least one virus depletion of step d) can also be carried out by a solvent detergent step, in which a virus depletion by solvent / detergent is achieved.
- the virus depletion can also be achieved by UV treatment and / or by thermal treatment.
- the at least one virus removal of step d) can be carried out in particular in a residence zone into which a segmented product stream can be introduced.
- all product-contacted elements used in steps (a) to (e) are germ-reduced by a suitable germ reduction method.
- the germ reduction method may preferably be selected from the group consisting of gamma irradiation, beta irradiation, autoclaving, ethylene oxide (ETO) treatment, ozone treatment (O 3 ), hydrogen peroxide treatment (H 2 O 2 ) and steam-in-solution. place (SIP) treatment.
- the articles and elements of the modules used in the process according to the invention which come into contact with the product stream are preferably germ-reducible and / or sterilizable, preferably autoclavable, gamma-irradiated, gassed with ethylene oxide (ETO) and treatable with ozone (O 3 ) , treatable with hydrogen peroxide (H 2 O 2 ) or treatable with a steam-in-place (SIP) treatment, allowing germ-reduced or even aseptic operation of the method of the invention.
- ETO ethylene oxide
- O 3 ozone
- SIP steam-in-place
- all product-contacting elements used starting from filtration step (b) are disposable articles or are used as disposable articles.
- Such used disposable articles can then also be referred to as "disposable” or “single-use” articles in the process according to the invention ("SU technology"). This improves the germ-reduced state of the process.
- all input fluids are filtered through a germ reduction filter, such as a Sartorius Sartoguard NF filter.
- all exits may preferably be protected by a germ barrier which prevents a re-growth of microorganisms.
- a Sartorius NF filter from Sartorius can also be used as a germ barrier here.
- An additional security of the germ barrier 11 can be achieved by a changeover of filters and / or waste line.
- Another measure to ensure germ-reduced conditions can be obtained by periodically sanitizing the waste line, preferably after filtration with e.g. NaOH solution can be achieved.
- Other methods would be UV irradiation and heat treatment.
- the modular method steps of the method according to the invention are preferably carried out in modules in a further embodiment, wherein the modules are interconnected.
- the modules may preferably be connected to one another by welding or by aseptic connectors.
- the modules eg the device "TC Welder" of the company Sartorius can be used.
- all the liquids, gases and solids used are germ-reduced in steps (a) to (e).
- the germ reduction preferably takes place by filtration through a filter having a pore size of preferably ⁇ 0.45 ⁇ m. In this case, preferably no in-process sterilization is carried out during the process.
- the seed reduction may also be carried out by filtration through a filter having a pore size of preferably ⁇ 0.20 ⁇ m.
- a degassing of all fluids is carried out before the chromatography step (c), which come to the at least two chromatography columns, wherein the degassing preferably by at least one bubble trap and / or by at least one hydrophobic microfiltration membrane Vacuum and / or by treatment with ultrasound and / or by bubbling with a poorly soluble gas, such as Helium takes place.
- hydrophobic microfiltration membrane via vacuum for maintaining the sterility in the continuous leadership of the invention Process and the system according to the invention preferred, since this has proven to be particularly advantageous compared to the bubble trap.
- the hydrophobic microfiltration membrane used can in particular be a MicroModule from Membrana.
- the particle-free fluid from step a) is subjected to at least one ultrafiltration against a microbicide-containing buffer.
- a microbicide-containing buffer By ultrafiltration, the nutrients contained in the fluid are exchanged for a microbicide-containing buffer, which microorganisms / germs should be removed from the growth base in the fluid. This further improves the germ reduction of the process.
- microbicide used here may preferably be selected from the group consisting of imidazole, benzoic acid, sorbic acid, para-hydroxybenzoic acid esters, sulfites, disulfites, azides, orthophenylphenol, nisin, natamycin, hexamethylenetetramine, dimethyldicarbonate, nitrites, nitrates , Acetic acid, ascorbic acid, isoascorbic acid, L-lactic acid, propionic acid, boric acid and lysozyme.
- the biopharmaceutical, biological macromolecular product is a protein or peptide selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant proteins and vaccines, preferably DNA and RNA vaccines.
- the chromatography columns and / or membrane adsorbers used in step c) may have any suitable binding principle, such as affinity of the product for a ligand, ionic interactions, metal chelate bonding, hydrophobic interactions or pure van der Waals forces.
- the first chromatographic step of the at least two chromatography steps may be affinity chromatography (eg, a ligand having affinity for the product, such as protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, IgG different recombinant protein which has an affinity for the product).
- affinity chromatography eg, a ligand having affinity for the product, such as protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, IgG different recombinant protein which has an affinity for the product.
- second chromatography step such as chromatography on ionic interactions.
- the process according to the invention is flexible here and can comprise in step c), depending on the degree of purity and concentration of the product to be achieved, any suitable chromatography principle in any order.
- the at least two chromatography columns and / or membrane adsorbers of step (c) comprise a ligand which is preferably selected from the group consisting of protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, and IgG different recombinant protein, which has an affinity for the product.
- the process of steps (a) to (e) has a runtime of at least 4 hours, preferably of at least 8 hours, preferably of at least 12 hours, preferably of at least 24 hours, more preferably of at least 48 Hours, more preferably at least 7 days, more preferably at least 4 weeks, and most preferably at least 8 weeks.
- At least one filtration step comprising at least one filter is carried out between steps a) to e) and / or thereafter.
- the simultaneous or downstream delivery of product into the new filter may e.g. done by a feed pump.
- the filter change switching from a used to a new filter element
- the filter venting proves to be problematic in this case, but it is necessary and must be carried out manually in the filters currently available.
- the filter of a germ reduction method by possibly ETO. Subjected to autoclaving or gamma irradiation and then connected to the process. Thereafter, the filter can be filled on the unfiltrate side, while the vent valve is open, which must be closed after successful venting, so that the actual filtration can be performed. In a batch process, a strong germ-reduced handling on the unfiltrate side is not required because it arrives in the short periods only germ-free as possible filtrate.
- vent valve For the automatic filter ventilation, it is advantageous to avoid the rotational movement of the vent valve during commissioning, so that the venting can be performed only by simple measures.
- the vent valve can now be modified so that it is continuous even in the closed state, but still reliably seals to the environment.
- the valve is thereby in the safe state "closed", which is characterized by a tight fit.
- the state "open” is usually not clearly defined, since the valve body in the guide shakes strong and allows a shift of the limit.
- This arrangement is now preferably germ-reduced by ETO, gamma irradiation, autoclaving, or ozone (O 3 ) treatment or by hydrogen peroxide (H 2 O 2 ) treatment.
- ETO gamma irradiation
- autoclaving or ozone (O 3 ) treatment
- H 2 O 2 hydrogen peroxide
- the process plant blocks the flow of production through a valve on the filtrate side, so that there is a pressure increase in front of the filter. This is detected by a suitable sensor. If a certain pressure threshold is exceeded, the breather squeeze valve is closed and the valve on the filtrate side is opened. In this procedure, the filter can be automatically modified without manual intervention with a modified vent valve.
- This automatic filter change is in FIG. 2 by way of example, where the working principle of the filtration step of the method and the installation is schematically illustrated by replacing a spent filter 17 by welding a new filter 16.
- the final purified biopharmaceutical biological macromolecular products from the heterogeneous cell culture-fluid mixture can be filtered one final time at the end of the process by a final filtration, preferably through a filter having a pore size of 0.2 ⁇ m.
- the final filtration can be gamma-irradiated, for example Sartopore 2 capsules (Midicap size 7, 0.05m 2 ) into a gamma-irradiated 5L GE ReadCircuit Bag. If the fill level of the final bag is sufficient then this bag can be welded off and a new bag welded to the process.
- the at least one chromatography module may also comprise more than two chromatography columns and / or membrane adsorbers, e.g. three or four chromatography columns and / or membrane adsorbers.
- all of the modules (a) to (d) used and germinated with product contact elements by a germ reduction method wherein the germ reduction method is preferably selected from the group consisting of gamma irradiation, beta irradiation, autoclaving , Ethylene oxide (ETO) treatment, ozone treatment (O 3 ), hydrogen peroxide treatment (H 2 O 2 ) and steam-in-place (SIP) treatment.
- the germ reduction method is preferably selected from the group consisting of gamma irradiation, beta irradiation, autoclaving , Ethylene oxide (ETO) treatment, ozone treatment (O 3 ), hydrogen peroxide treatment (H 2 O 2 ) and steam-in-place (SIP) treatment.
- the modular system itself, as well as the elements of the modules that come into contact with product flow, germ-reducible and / or sterilizable, preferably autoclavable, gamma-irradiated, gassed with ethylene oxide (ETO), with ozone (O 3 ) treatable with hydrogen peroxide (H 2 O 2 ) treatable or with a steam-in-place (SIP) treatment treatable, which allows a germ-free or even aseptic operation of the modular system according to the invention.
- ETO ethylene oxide
- O 3 ozone
- H 2 O 2 hydrogen peroxide
- SIP steam-in-place
- all product-contacted articles used in the modules (a) to (d) are disposable articles or are used as disposable articles.
- the modules are preferably connected to one another by welding or by aseptic connectors.
- aseptic "ReadyMate” connectors from GE are preferably aseptic connectors used in the modular system according to the invention.
- Prefabricated disposable articles (“disposables", “ready-to-use") are preferably used as gamma-irradiated elements.
- all input fluids pass through a germ reduction filter, wherein preferably all exits are protected by a germ barrier which prevents regress growth.
- the final purified biopharmaceutical biological macromolecular products from the heterogeneous cell culture fluid mixture can be filtered one final time at the end of the modular unit through a final filtration, preferably through a filter having a pore size of 0.2 ⁇ m.
- Final filtration can be done, for example, via gamma-irradiated Sartopore 2 capsules (Midicap size 7, 0.05m 2 ) into a gamma-irradiated 5L GE ReadCircuit Bag. If the fill level of the final bag is sufficient then this bag can be welded off and a new bag welded to the process.
- Table 1 shows the mean flow rates and antibody concentrations of in FIG. 1 positions shown.
- a 10 L perfusion reactor was used for the continuous production of a monoclonal antibody IgG.
- the viable cell density in the stationary state was 60-70 million cells / mL.
- the titer was ⁇ 115 mg / L.
- the production was operated with two parallel perfusion reactors for 28 days.
- the product was continuously removed via a slant plate separator (settler), which retained most of the cells.
- Fig. 2 shows how a closed germ-free process was realized here.
- Both the filters and the hose assembly were gamma irradiated.
- the input and output lines were connected via aseptic connectors to gamma-irradiated bags (GE ReadyCircuit 1 L), which served as equalization volumes for fluctuating flow rates.
- GE ReadyCircuit 1 L gamma-irradiated bags
- To deaerate the filters were coupled to hydrophobic 0.2 micron air filter whereby the module was closed in the sense of the invention ( Fig.2 ).
- the air filter was either an Emflon II from Pall Corp. or a Midisart2000 of the company Sartorius Stedim.
- vent valves were modified in such a way that they were consistent even when closed, but still reliably sealed to the environment.
- the inner sealing ring of the vent valve was removed and the valve was closed before the gamma irradiation. This valve was additionally secured against opening. As a result, the valve was in a safe condition "closed", which was characterized by a tight fit.
- the vent valve was connected to the air filter via a hose. Between the vent valve and the air filter, the hose was inserted into a pinch valve. The filter was filled until liquid entered the air filter through the vent valve and hose. The hydrophobic vent filter then blocked the liquid.
- the process plant blocked the flow of production through a valve on the filtrate side, causing the pressure in front of the filter to rise. This pressure increase was detected by a Pendotech pressure sensor. If a threshold of 0.5 bar was exceeded, the breather valve pinch valve was closed and the valve on the filtrate side opened.
- the filtrate from i) cell clarification filtration was first passed through an ultrafiltration hollow fiber membrane (GE Healthcare Ready to Process, 0.2m 2 , gamma-irradiated). continuously concentrated by a factor of 10.
- the circulation pump was a disposable QuattroFlow 1200 SU pump whose pump head was integrated into the hose assembly before the gamma radiation.
- the media components of the concentrated product were then exchanged with a 50 mM imidazole / NaCl buffer over a Gambro Revaclear 300 dialysis membrane.
- the module is supplied sterile packaged by the manufacturer and connected to the gamma irradiated hose assembly under a sterile bench.
- the permeate from the concentration and the media-laden waste stream were fed into a gamma-irradiated 200L Sartorius Flexboy.
- the replacement of the Flexboys was carried out by welding with a Sartorius welding machine.
- the product was continuously filtered with gamma-irradiated Sartoguard NF filters (MaxiCap Size 8) in a 200L Flexboy before filling. Construction and operation was analogous to B. Downstream DSP-1 i) cell clarification filtration.
- Mabselect Sure was used as Prot-A resin to isolate the IgG.
- the IgG was concentrated up to a factor of 10 and most of the contaminants were removed.
- a continuous BioSMB plant from Tarpon Biosystems, Inc. was used with 12 columns (ID 16 mm, L 80 mm) with 8 columns in the loading zone (2 columns in series and 4 parallel series). The entire flow path, including the columns, was rendered germ-free by sanitization or gamma irradiation.
- the loading per cycle was 32 column volumes per column.
- As buffer were acetate buffer with different Molarity, pH and conductivities used.
- Virus inactivation and neutralization consisted of three modules and was between capture chromatography and a 0.2 ⁇ m filtration: (a) a homogenization loop with a peristaltic loop hose pump M0502 (b) shown schematically as a coiled tubing (c) a neutralizing bag in the the pH could be adjusted to 7.5.
- the modules were made according to the welds in Figure 3 individually prefabricated and gamma-irradiated, with the ends each welded shut.
- pH probes here pH0501 and pH0502
- pH probe sections were autoclaved. pH probe sections were then sealed in assemblies. Bags were shown connected via aseptic GE Ready Mate ® connectors. The connections to the protein A eluate line and filtration module were initially sealed and then welded to the respective modules.
- Proteins were able to precipitate after a pH shift, whereby the precipitated protein was filtered off.
- the product from the above 0.2 ⁇ m filtration was passed through two chromatographic steps by first using four sequentially (4-PCC) 2.5ml Capto Adhere (2.5ml GE) and then two 20ml alternating anion exchangers (Pall Hypercel StarAX). purified. ProtA leachables, DNA, HCP and aggregates were purified.
- the two chromatography steps were a gammabestrahles Bag (GE Circuit Ready ® 1 L) connected with each other, in which the conductivity were adjusted according to the requirement of the anion exchanger on 7,5mS / cm by adding water.
- the entire river route including the columns was sanitized or gamma-irradiated.
- the loading per cycle was 50 column volumes per column.
- the waste stream was directed into a gamma-irradiated 200L Sartorius Flexboy.
- the replacement of the Flexboys was carried out by welding with a Sartorius welding machine.
- the product stream was again collected in a gamma-irradiated product bag (GE ReadyCircuit 1 L).
- the product solution was first prefiltered from the product bag of the Polish Chromatography via a 0.1 ⁇ m capsule (Sartopore2, MidiCap size 9, 0.2 m 2 ). Implementation and setup were analogous to the B. Downstream DSP-1 i) cell clarification filtration.
- the output line from the prefiltration was connected directly via a peristaltic pump to the input of virus filtration from C. Downstream DSP-II via welding. Otherwise, the setup and operation of virus filtration from C. Downstream DSP-II was analogous to C. Downstream DSP-II 0.2 ⁇ m filtration. However, the virus filter used was a Virosart CPV filter (MidiCap size 9, 0.2 m 2 ), which was rinsed and autoclaved according to the manufacturer's instructions. Again, the filters were in the assembly shrink wrapped. The product stream was pumped again into a gamma-irradiated product bag (GE ReadyCircuit 1 L).
- the final concentration and rebuffering was analogous to the above B. DSP-I ii) concentration and rebuffering and differed only in that an autoclaved UV cell was involved in monitoring the product concentration in the concentration loop. The rebuffering was also carried out analogously to the above B. DSP-I ii) concentration and rebuffering, here a 50mM phosphate buffer pH 7.5 were used. The product stream was pumped again into a gamma-irradiated product bag (GE ReadyCircuit 1 L).
- a regularly run time of the method according to the invention was 3 days without germ growth, wherein the chromatography columns were sanitized by a 40% isopropanol + 0.5 M NaOH. At run times of the inventive method of more than 3 days, the chromatography columns were gamma-irradiated.
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Die Erfindung eines Verfahrens zur kontinuierlichen, keimreduzierten Produktion und/oder Aufbereitung eines biopharmazeutischen, biologischen makromolekularen Produktes aus einer heterogenen Zellkultur-Fluid-Mischung zur Verfügung, umfassend die Schritte: (a) Bereitstellen eines partikel-freien Fluids aus einer heterogenen Zellkultur-Fluid-Mischung, welche das Produkt enthält, in Form eines Produktstroms, (b) mindestens eine Filtration, wobei ein Filtrat erhalten wird, (c) wenigstens zwei Chromatographieschritte zur Reinigung des Produktes, (d) mindestens eine Virenabreicherung, (e) mindestens eine Ultrafiltration und/oder mindestens eine Diafiltration des Produktstroms der Schritte (b), (c), und/oder (d), dadurch gekennzeichnet, dass die wenigstens zwei Chromatographieschritte aus (c) eine Reinigung über wenigstens jeweils zwei Chromatographiesäulen und/oder Membranadsorber umfasst und, dass das Verfahren geschlossen und modular durchgeführt wird. Die Erfindung stellt weiterhin eine entsprechende modulare Anlage zur Durchführung dieses Verfahrens zur Verfügung.The invention of a method for the continuous, germ-reduced production and / or preparation of a biopharmaceutical, biological macromolecular product from a heterogeneous cell culture fluid mixture, comprising the steps: (a) providing a particle-free fluid from a heterogeneous cell culture fluid (B) at least one filtration to obtain a filtrate, (c) at least two chromatographic steps to purify the product, (d) at least one virus depletion, (e) at least one ultrafiltration, and or at least one diafiltration of the product stream of steps (b), (c), and / or (d), characterized in that the at least two chromatographic steps of (c) comprises a purification via at least two chromatography columns and / or membrane adsorbers, and that the process is closed and modular. The invention further provides a corresponding modular system for carrying out this method.
Description
Die Erfindung betrifft eine modulare Anlage und ein Verfahren zur kontinuierlichen, keimreduzierten Produktion und/oder Aufbereitung eines Produktes aus einer heterogenen Zellkultur-Fluid-Mischung.The invention relates to a modular system and a method for the continuous, germ-reduced production and / or preparation of a product from a heterogeneous cell culture fluid mixture.
Gewöhnlich werden Proteine in der biotechnologischen Herstellung im Batch aufgereinigt (englisch batch: "Stapel"). Das bedeutet, dass die einzelnen Produktionszyklen chargenweise, diskontinuierlich gehandhabt werden, wobei nach dem Abschluss eines Produktionszyklus das Produkt alles Ganzes entnommen wird. Für eine erneute Produktion muss dann separat ein neuere Produktzyklus bzw. Batch gestartet werden.Usually proteins are purified in the biotechnological production in batch (English batch : "stack"). This means that the individual production cycles are handled in batches, discontinuously, and after the completion of a production cycle, the product is removed from the whole. For a new production then a newer product cycle or batch must be started separately.
In den letzten Jahren hat sich nun mehr und mehr gezeigt, dass eine kontinuierliche Verfahrensweise auch in der biotechnologischen Herstellung gefahren werden kann, wobei das Verfahren im Gegensatz zum Batch-Verfahren ohne Unterbrechungen laufen kann.In recent years, it has become more and more evident that a continuous procedure can also be used in biotechnological production, whereby the process can run without interruptions in contrast to the batch process.
Die stark regulierte pharmazeutische Produktion erfordert einen großen zeitlichen, technischen und personellen Aufwand für die Bereitstellung gereinigter und sterilisierter Bioreaktoren und Gewährleistung eines keimfreien Produkts. Um Kreuzkontaminationen bei einem Produktwechsel in einer Multi-Purpose-Anlage oder zwischen zwei Produktchargen sicher zu vermeiden, wird außer der Reinigung eine sehr aufwändige Reinigungsvalidierung benötigt, welche bei einer Prozessadaption ggf. wiederholt werden muss.
Dies gilt sowohl für das Upstream-Processing (USP), d.h. die Herstellung biologischer Produkte in Fermentern als auch für das Downstream-Processing (DSP), d. h. die Aufreinigung der Fermentationsprodukte.
Gerade bei der Fermentation ist eine keimfreie Umgebung für eine erfolgreiche Kultivierung essentiell. Zur Sterilisation von Batch- oder Fedbatch-Fermentern kommt in der Regel die SIP-Technik zum Einsatz (SIP = sterilization-in-place).
Der durch die Bereitstellungsprozeduren bedingte Nutzungsausfall der Reaktoren kann insbesondere bei kurzen Nutzungsperioden und häufigem Produktwechsel in der Größenordnung der Reaktorverfügbarkeit liegen. Betroffen sind im USP der biotechnologischen Produktion z. B. die Prozessschritte der Medienherstellung und Fermentation und im DSP das Solubilisieren, Einfrieren, Auftauen, pH-Adjustieren, Produkttrennung wie z. B. durch Chromatographie, Fällen, oder Kristallisieren, das Umpuffern und die Virusinaktivierung.Highly regulated pharmaceutical production requires a great deal of time, technical and human resources to provide purified and sterilized bioreactors and ensure a germ-free product. In order to reliably avoid cross-contamination during a product change in a multi-purpose system or between two product batches, a very expensive cleaning validation is required in addition to cleaning, which may need to be repeated during a process adaptation.
This applies both to the upstream processing (USP), ie the production of biological products in fermenters and for the downstream processing (DSP), ie the purification of the fermentation products.
Especially in fermentation, a germ-free environment is essential for successful cultivation. For the sterilization of batch or fed-batch fermenters, the SIP technique is usually used (SIP = sterilization-in-place).
The loss of use of the reactors due to the provisioning procedures may be on the order of the reactor availability, in particular in the case of short periods of use and frequent product changes. Affected in the USP of biotechnological production z. B. the process steps of media production and fermentation and DSP solubilization, freezing, thawing, pH adjustment, Product separation such. By chromatography, precipitation, or crystallization, rebuffering, and virus inactivation.
Die regulatorischen Anforderungen sind dabei im Downstream-Bereich eine keimarme Prozessführung. Daher ist eine sterile Fahrweise im Falle des Batch-Betriebes nicht gefordert. In einem kontinuierlichen Verfahren jedoch wird die Reinigung des Proteins über einen längeren Zeitraum möglichst ohne Reinigungsschritte betrieben. Dies geschieht bevorzugt ohne Sterilisationsschritte während der Reinigung. Dabei ist aber das Verkeimungsrisiko um ein vielfaches höher als bei einem reinen Batchbetrieb.The regulatory requirements in the downstream area are low-germ litigation. Therefore, a sterile procedure in the case of batch operation is not required. In a continuous process, however, the purification of the protein is operated over a longer period of time as possible without purification steps. This is preferably done without sterilization steps during cleaning. However, the risk of contamination is many times higher than in a pure batch mode.
Ein kontinuierliches Verfahren meint im Rahmen der Erfindung jeden Prozess zur Durchführung von mindestens zwei Prozessschritten in Serie, in dem der Ausgangsstrom eines vorgeschalteten Schrittes in einen nachgeschalteten Schritt befördert wird. Der nachgeschaltete Schritt beginnt die Bearbeitung des Produktstroms, bevor der vorgeschaltete Schritt abgeschlossen ist. Üblicherweise wird in einem kontinuierlichen Verfahren immer ein Teil des Produktstroms in der Produktionsanlage befördert und wird als "kontinuierlicher Produktstrom" bezeichnet. Entsprechend bedeutet eine kontinuierliche Beförderung oder Transfer eines Produktstroms von einer vorgeschalteten Unit in eine nachgeschaltete Unit, dass die nachgeschaltete Unit bereits arbeitet, bevor die vorgeschaltete Unit außer Betrieb genommen wird, d.h. dass zwei nacheinander geschaltete Units den Produktstrom gleichzeitig prozessieren, der sie durchfließt.A continuous process in the context of the invention means any process for carrying out at least two process steps in series, in which the output stream of an upstream step is conveyed into a downstream step. The downstream step begins processing the product stream before the upstream step is completed. Usually, in a continuous process, a part of the product stream is always carried in the production plant and becomes referred to as "continuous product stream". Correspondingly, a continuous transport or transfer of a product stream from an upstream unit to a downstream unit means that the downstream unit is already operating before the upstream unit is taken out of service, ie, two successive units simultaneously process the product stream that flows through it.
Der Begriff "keimreduziert" meint im Rahmen der Erfindung einen Zustand reduzierter Keimzahl, also einer Zahl an Mikroorganismen pro Flächen- oder Volumeneinheit von nahezu Null, der durch eine geeignete Keimreduktionsmethode erreichbar ist, wobei diese Keimreduktionsmethode ausgewählt sein kann aus Gamma-Bestrahlung, Beta-Bestrahlung, Autoklavieren, Ethylenoxid (ETO)-Behandlung und "steam-in-place" (SIP)-Behandlung.The term "germ-reduced" in the context of the invention means a state of reduced germ count, ie a number of microorganisms per unit area or volume unit of almost zero, which can be achieved by a suitable germ reduction method, this germ reduction method can be selected from gamma irradiation, beta Irradiation, autoclaving, ethylene oxide (ETO) treatment and steam-in-place (SIP) treatment.
Der Begriff "Einwegartikel" bedeutet im Rahmen der Erfindung, dass die betreffenden produkt-berührten Teile, insbesondere Apparaturen, Behältern, Filter und Verbindungselemente, zum einmaligen Gebrauch mit anschließendem Wegwerfen geeignet sind (Englisch "disposable"), wobei diese Behälter sowohl aus Kunststoff als auch aus Metall sein können. Im Rahmen der Erfindung umfasst der Begriff auch Mehrwegartikel, etwa aus Stahl, die im erfindungsgemäßen Verfahren nur einmal verwendet werden und dann im Verfahren nicht mehr zum Einsatz kommen. Diese Mehrwegartikel, z.B. aus Stahl, werden dann im Rahmen der Erfindung auch "als Einwegartikel benutzte" Gegenstände bezeichnet. Solche eingesetzten Einwegartikel können dann im erfindungsgemäßen Verfahren auch als "disposabel" bzw. "Single-Use"- Artikel bezeichnet werden ("SU Technologie"). Dadurch wird der keimreduzierte Zustand des erfindungsgemäßen Verfahrens und modularer Anlage noch weiter verbessert.In the context of the invention, the term "disposable article" means that the product-contacted parts in question, in particular apparatuses, containers, filters and connecting elements, are disposable with disposable use, these containers being made of plastic as well as plastic also be made of metal. In the context of the invention, the term also includes reusable articles, for example of steel, which are used only once in the process according to the invention and are then no longer used in the process. These reusable articles, e.g. made of steel, are then referred to in the context of the invention "used as disposable" items. Such used disposable articles can then also be referred to as "disposable" or "single-use" articles in the process according to the invention ("SU technology"). As a result, the germ-reduced state of the method according to the invention and modular system is further improved.
Der Begriff "Produktstrom" meint im Rahmen der Erfindung das partikel-freie Fluid aus einer heterogenen Zellkultur-Fluid-Mischung, welche das Produkt enthält, sowie das Ergebnis jeder anderen Verfahrensschritte des erfindungsgemäßen Verfahrens, also den Produktstrom nach Filtration, nach Chromatographie, nach Virenabreicherung, nach Ultrafiltration, nach Diafiltration, oder nach weiteren Schritten des erfindungsgemäßen Verfahrens, wobei diese Produktströme dann unterschiedliche Konzentrationen und Reinheitsgrade aufweisen können.The term "product stream" in the context of the invention means the particle-free fluid from a heterogeneous cell culture fluid mixture which contains the product, as well as the result of every other process steps of the process according to the invention, ie the product stream after filtration, after chromatography, after virus depletion after ultrafiltration, after diafiltration, or after further steps of the process according to the invention, these product streams then being able to have different concentrations and degrees of purity.
Der Begriff "Virenabreicherung" meint im Rahmen der Erfindung eine Verringerung der Konzentration an aktiven Viren pro Volumeneinheit des zu behandelnden Fluids, bis hin zur vollständigen Inaktivierung und/oder Entfernung der in dem zu behandelnden Fluid enthaltenen Viren.The term "virus depletion" in the context of the invention means a reduction in the concentration of active viruses per unit volume of the fluid to be treated, to the complete inactivation and / or removal of the viruses contained in the fluid to be treated.
Der Begriff "Mikrobizid" meint im Rahmen der Erfindung einen Stoff, welcher das Wachstum von Mikroorganismen verlangsamen oder ganz hemmen kann, wobei dieses Mikrobizid in Form eines mikrobizid-haltigen Puffers, insbesondere im Rahmen des erfindungsgemäßen Verfahrens während einer Ultrafiltration verwendet werden kann.The term "microbicide" in the context of the invention means a substance which can slow or completely inhibit the growth of microorganisms, it being possible to use this microbicide in the form of a microbicidal buffer, in particular within the scope of the process according to the invention during ultrafiltration.
Der Begriff "Bubble-Trap" meint im Rahmen der Erfindung eine Vorrichtung zum Auffangen von Gasblasen, wobei dabei das betreffende Fluid entgast wird.The term "bubble trap" in the context of the invention means a device for collecting gas bubbles, wherein the relevant fluid is degassed.
Der Begriff "modular" meint im Rahmen der Erfindung, dass die einzelnen Schritte des erfindungsgemäße Verfahrens in separaten, miteinander verbundenen Modulen durchgeführt werden können, wobei die Module vorkonfiguriert und keimreduziert sind und geschlossen, in unterschiedlichen Kombinationen miteinander verschaltet werden können.The term "modular" means within the scope of the invention that the individual steps of the method according to the invention can be carried out in separate, interconnected modules, wherein the modules are preconfigured and germ-reduced and can be connected together in different combinations.
Der Begriff "modulare Anlage" meint im Rahmen der Erfindung eine Serie von miteinander verbundenen Modulen ("Units") zur Durchführung von mindestens zwei Downstream- und / oder Upstream-Schritten, in denen ein Fluid ("Produktstrom") befördert werden kann. Erfindungsgemäß sind die Units zur kontinuierlichen Durchführung eines Schrittes geeignet und können mit einem kontinuierlichen Fluidstrom ("Produktstrom") betrieben werden. Die einzelnen Module der "modularen Anlage" können dabei in jeder Kombination miteinander verschaltet werden. Beispiele für Module im Sinne der Erfindung sind das Filtrationsmodul 2, das Chromatographiemodul 3, das Ultrafiltrationsmodul 6, das Diafiltrationsmodul 7 und das Dialysemodul 8.The term "modular plant" in the context of the invention means a series of interconnected modules ("units") for carrying out at least two downstream and / or upstream steps in which a fluid ("product stream") can be conveyed. According to the invention, the units are suitable for the continuous performance of one step and can be operated with a continuous fluid flow ("product flow"). The individual modules of the "modular system" can be interconnected in any combination. Examples of modules within the meaning of the invention are the
Der Begriff "geschlossen" bedeutet im Rahmen der Erfindung die Fahrweise des erfindungsgemäßen Verfahrens und der erfindungsgemäßen modularen Anlage, welche so gefahren werden, dass das Produkt, welches mit diesem Verfahren und dieser modularen Anlage produziert und/oder aufbereitet wird, nicht an die Raumumgebung exponiert wird. In das erfindungsgemäße geschlossene Verfahren und die entsprechende erfindungsgemäße geschlossene modulare Anlage können von außen Materialien, Gegenstände, Puffer und dergleichen zugegeben werden, wobei aber diese Zugabe in einer solchen Weise erfolgt, dass eine Exposition des produzierten und/oder aufbereiteten Produktes an die Raumumgebung vermieden wird.The term "closed" in the context of the invention means the mode of operation of the method according to the invention and the modular system according to the invention, which are operated such that the product which is produced and / or processed with this method and this modular system is not exposed to the room environment becomes. In the closed method according to the invention and the corresponding closed modular system according to the invention, materials, objects, buffers and the like can be added externally, but this addition takes place in such a way that exposure of the produced and / or processed product to the room environment is avoided ,
Die im Stand der Technik bekannten, üblichen Verfahren weisen eine Reihe von Nachteilen auf, welche im Folgenden behandelt werden.The conventional processes known in the art have a number of disadvantages, which will be dealt with below.
Bekannte Verfahren zur Herstellung von biopharmazeutischen und biologischen Produkten umfassen üblicherweise folgende Produktionsschritte, die miteinander verschaltet werden:
- 1. Perfusionskultur
- 2. Zellrückhaltesystem,
- 3. Zellabtrennung
- 4. Puffer- bzw. Medienaustausch bevorzugt mit Konzentrierung
- 5. BioBurden-Reduzierung bevorzugt durch Sterilfiltration
- 6. Capture Chromatographie
- 1. perfusion culture
- 2. cell restraint system,
- 3. cell separation
- 4. Buffer or media exchange preferably with concentration
- 5. BioBurden reduction preferably by sterile filtration
- 6. Capture chromatography
Üblicherweise werden weitere Schritte zur weiteren Reinigung des Produktstroms durchgeführt, insbesondere:
- 7. Virusinaktivierung
- 8. Neutralisation, und
- 9. Optional eine weitere Tiefenfiltration, BioBurden-Reduzierung (Sterilfiltration).
- 7. Virus inactivation
- 8. Neutralization, and
- 9. Optional further depth filtration, BioBurden reduction (sterile filtration).
Angesichts der hohen Qualitätsstandards bei der Herstellung von Biopharmazeutika folgen üblicherweise darüber hinaus folgende Schritte:
- 10. Chromatographische Zwischen- und Feinreinigung
- 11. BioBurden Reduzierung z. B. Sterilfiltration
- 12. Virenfiltration
- 13. Pufferaustausch und bevorzugt Konzentrierung, und
- 14. Sterilfiltration.
- 10. Chromatographic intermediate and fine cleaning
- 11. BioBurden reduction z. B. Sterile filtration
- 12. Virus filtration
- 13. buffer exchange and preferably concentration, and
- 14. Sterile filtration.
Bei der oben beschriebenen Herstellung stellen Zellen in einem Fermenter mit Nährstofflösung ein biologisches Produkt her, etwa ein Protein, beispielsweise ein therapeutisches Protein. Die Nährstofflösung ist dabei auch ein ideales Wachstumsmedium für Mikroorganismen, wie Bakterien und Sporen, woraus sich ein Problem hinsichtlich des ungewünschten Wachstums solcher Mikroorganismen ergibt. Dieses ungewünschte Wachstum von Mikroorganismen wird besonders bei längeren Laufzeiten ein Problem, weil die Nährstofflösung mit ansteigender Laufzeit des Verfahrens mehr und mehr kontaminiert wird, bis hin zu einem exponentiellen Wachstum von Mikroorganismen und damit Totalverlust der betreffenden Charge an hergestelltem biologischen Produkt.In the preparation described above, cells in a fermentor with nutrient solution produce a biological product, such as a protein, for example, a therapeutic protein. The nutrient solution is also an ideal growth medium for microorganisms, such as bacteria and spores, resulting in a problem with regard to the unwanted growth of such microorganisms. This undesirable growth of microorganisms becomes a problem, especially with longer maturities, because the nutrient solution becomes more and more contaminated with increasing runtime of the process, up to an exponential growth of microorganisms and thus total loss of the respective batch of biological product produced.
Um der Forderung an ein schnelles und flexibles Neubeschicken der Produktionsanlage unter Wahrung maximaler Sauberkeit und Keimfreiheit gerecht zu werden, erfreuen sich auf dem Markt Konzepte für eine kontinuierliche Produktion bevorzugt mit Einwegtechnologie eines ständig wachsenden Interesses.In order to meet the demand for a fast and flexible new shipment of the production plant while maintaining maximum cleanliness and sterility, the market enjoys concepts for continuous production, preferably with disposable technology of ever increasing interest.
Für längere Laufzeiten eines solchen Verfahrens von mehreren Stunden über Tage bis Wochen reichen aber gewöhnliche Maßnahmen zur Sanitisierung nicht, etwa die üblichen "Clean-in-Place" (CIP)-Maßnahmen, wie z.B. Sanitisierung durch 1M NaOH. Bei Laufzeiten ab mehreren Stunden haben solche gewöhnlichen Verfahren und Anlagen daher den Nachteil, dass sie hinsichtlich möglicher Kontamination und/oder möglichen Keimwachstums sehr anfällig sind.However, for longer run times of such a process, from several hours over days to weeks, ordinary sanitizing measures will not suffice, such as the usual "clean-in-place" (CIP) measures, such as, e.g. Sanitization by 1M NaOH. With maturities of several hours, such common processes and plants therefore have the disadvantage that they are very susceptible to possible contamination and / or possible germ growth.
Es bestand daher ein Bedarf an einem Verfahren zur kontinuierlichen Reinigung eines Produktes aus einer heterogenen Zellkultur-Fluid-Mischung, welches durch seine weitgehende Keimarmut eine kontinuierliche Fahrweise von bis zu mehreren Wochen ermöglicht.There was therefore a need for a process for the continuous purification of a product from a heterogeneous cell culture-fluid mixture, which allows a continuous operation of up to several weeks due to its extensive germ reduction.
Es war daher die Aufgabe der vorliegenden Erfindung, ein Verfahren und eine entsprechende Anlage zu entwickeln, mit dem ein Produkt, etwa ein Protein, kontinuierlich über einen Zeitraum von mehreren Stunden bis hin zu mehreren Wochen gereinigt werden kann.It was therefore the object of the present invention to develop a method and a corresponding system with which a product, for example a protein, can be purified continuously over a period of several hours to several weeks.
Die Erfindung löst diese Aufgabe durch Bereitstellen eines Verfahrens zur kontinuierlichen, keimreduzierten Produktion und/oder Aufbereitung eines biopharmazeutischen, biologischen makromolekularen Produktes aus einer heterogenen Zellkultur-Fluid-Mischung, umfassend die Schritte:
- (a) Bereitstellen eines partikel-freien Fluids aus einer heterogenen Zellkultur-Fluid-Mischung, welche das Produkt enthält, in Form eines Produktstroms,
- (b) mindestens eine Filtration, wobei ein Filtrat erhalten wird,
- (c) wenigstens zwei Chromatographieschritte zur Reinigung des Produktes,
- (d) mindestens eine Virenabreicherung,
- (e) mindestens eine Ultrafiltration und/oder mindestens eine Diafiltration des Produktstroms der Schritte (b), (c), und/oder (d),
- (a) providing a particle-free fluid from a heterogeneous cell culture fluid mixture containing the product in the form of a product stream,
- (b) at least one filtration, whereby a filtrate is obtained,
- (c) at least two chromatographic steps to purify the product,
- (d) at least one virus depletion,
- (e) at least one ultrafiltration and / or at least one diafiltration of the product stream of steps (b), (c), and / or (d),
Die Grundprinzipien des erfindungsgemäßen Verfahrens beruhen auf seinen vier Kernprinzipien und damit Kernmerkmalen:
- 1. kontinuierliche
- 2. keimreduzierte
- 3. geschlossene, und
- 4. modulare Produktion eines biopharmazeutischen, biologischen makromolekularen Produktes.
- 1. continuous
- 2. germ-reduced
- 3. closed, and
- 4. Modular production of a biopharmaceutical, biological macromolecular product.
Das in Schritt a) bereitgestellte partikel-freie Fluid aus einer heterogenen Zellkultur-Fluid-Mischung kann bevorzugt aus einem kontinuierlichen Perfusions- und Fermentationsprozesses stammen, etwa eine Zellkultur bzw. Gewebekultur, oder ein Perfusionsreaktor. Dabei können sogar mehr als ein Perfusionsreaktor parallel betrieben werden, etwa zwei Perfusionsreaktoren.The particle-free fluid from a heterogeneous cell culture-fluid mixture provided in step a) can preferably originate from a continuous perfusion and fermentation process, for example a cell culture or a perfusion reactor. In this case, even more than one perfusion reactor can be operated in parallel, for example two perfusion reactors.
Das Fluid kann zunächst durch geeignete Zellrückhaltesysteme, etwa einen Schrägplattenabscheider (Settler) kontinuierlich abgeführt werden, durch den ein Großteil der Zellen zurückgehalten werden kann. Durch nachfolgende Filtrations- und/oder Zentrifugationsschritte oder andere geeignete Trennverfahren kann dann das Fluid von im Fluid enthaltenden Partikeln befreit werden, wodurch ein partikel-freies Fluid erhalten wird, in welchem sich das biopharmazeutische, biologische makromolekulare Produkt befindet.The fluid may be initially removed by suitable cell retention systems, such as a slant plate separator (settler), through which a majority of the cells can be retained. Subsequent filtration and / or centrifugation steps or other suitable separation processes may then free the fluid from particles contained in the fluid, thereby yielding a particle-free fluid in which the biopharmaceutical biological macromolecular product resides.
Der Filtrationsschritt (b) kann z.B. eine Filtration des nach Schritt (a) erhaltenen partikel-freien Fluids sein, wobei ein Filtrat erhalten wird. Das erfindungsgemäße Verfahren kann aber auch weitere Filtrationsschritte an geeigneten Stellen des Verfahrens umfassen.
Der Filtrationsschritt b) kann durch geeignete Filterverfahren erfolgen, z.B. einen 0,2µm Filter, oder mehrerer parallel betriebene Filter. Ein geeigneter Filter für den Filtrationsschritt ist beispielsweise ein Sartoguard NF 0,2µm Filter, von dem mehrere parallel betrieben werden können.
In einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens umfasst der Filtrationsschritt (b) einen Tiefenfilter mit einer zusätzlichen Möglichkeit der Abreicherung von Kontaminanten wie DNA, ProtA, HCP. Dies können Tiefenfilter mit ausreichendem Zetapotential (Zetapor 3M, Posidyne, Pall) oder Tiefenfilter mit Aktivkohle (Millistak MerckMillipore) sein.The filtration step (b) may be, for example, a filtration of the particle-free fluid obtained after step (a), whereby a filtrate is obtained. However, the process according to the invention can also comprise further filtration steps at suitable points of the process.
The filtration step b) can be carried out by suitable filtering methods, eg a 0.2 μm filter or several filters operated in parallel. A suitable filter for the filtration step is for example a Sartoguard NF 0.2μm filter, several of which can be operated in parallel.
In a further embodiment of the method according to the invention, the filtration step (b) comprises a depth filter with an additional possibility of depleting contaminants such as DNA, ProtA, HCP. These may be depth filters with sufficient zeta potential (Zetapor 3M, Posidyne, Pall) or activated carbon activated carbon filters (Millistak Merck Millipore).
Die wenigstens zwei Chromatographieschritte zur Reinigung des Produktes des Schrittes c) umfassen eine Reinigung über wenigstens jeweils zwei Chromatographiesäulen und/oder Membranadsorber. Dabei können die Chromatographiesäulen und/oder Membranadsorber jedes geeignete Bindungsprinzip aufweisen, etwa Affiniät des Produktes zu einem Liganden, ionische Wechselwirkungen, Metalchelat-Bindung, hydrophobe Interaktionen oder van-der-Waals-Kräfte. Beispielsweise kann der erster Chromatographieschritt der wenigstens zwei Chromatographieschritte eine Affinitätschromatographie sein (z.B. ein Ligand mit Affinität zum Produkt, wie z.B. Protein A, Protein G, Protein L, IgM, IgG und ein von Protein A, Protein G und Protein L, IgM, IgG unterschiedlichen rekombinanten Protein, welches eine Affinität für das Produkt hat). Danach folgt dann ein weiterer (zweiter) Chromatographieschritt, etwa eine Chromatographie über ionische Wechselwirkungen.The at least two chromatographic steps for purifying the product of step c) comprise purification via at least two chromatography columns and / or membrane adsorbers. The chromatographic columns and / or membrane adsorbers may have any suitable binding principle, such as affinity of the product for a ligand, ionic interactions, metal chelate bonding, hydrophobic interactions or van der Waals forces. For example, the first chromatographic step of the at least two chromatography steps may be affinity chromatography (eg, a ligand having affinity for the product, such as protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, IgG different recombinant protein which has an affinity for the product). This is followed by another (second) chromatography step, such as chromatography on ionic interactions.
Das erfindungsgemäße Verfahren ist hier flexibel und kann in Schritt c) je nach zu erzielendem Reinheitsgrad und Konzentration des Produktes jedes geeignete Chromatographieprinzip in jeder Reihenfolge umfassen.The process according to the invention is flexible here and can comprise in step c), depending on the degree of purity and concentration of the product to be achieved, any suitable chromatography principle in any order.
Der technische Effekt der Verwendung von wenigstens zwei Chromatographieschritten über wenigstens jeweils zwei Chromatographiesäulen und/oder Membranadsorbern gemäß Schritt c), besteht darin, dass in der Regel ein einzelner Chromatographieschritt keine ausreichende Aufreinigung der Kontaminaten wie z.B. Host Cell Impurities, Aggregate, DNA, Protein A usw. gewährleisten kann.
Außerdem wird dadurch eine kontinuierliche Produktion in einem Chromatographieschritt ermöglicht, da mindestens eine Chromatographiesäule und/oder Membranadsorber mit ungereinigtem Produkt beladen werden kann, während mindestens eine andere Chromatographiesäule und/oder Membranadsorber regeneriert bzw. eluiert werden kann, wodurch eine kontinuierliche und effektive Fahrweise des Verfahrens erzielt werden kann.The technical effect of using at least two chromatographic steps over at least two chromatography columns and / or membrane adsorbers according to step c), is that usually a single chromatography step does not sufficiently clean the contaminants such as host cell impurities, aggregates, DNA, protein A etc. can ensure.
In addition, this allows continuous production in a chromatographic step, since at least one chromatography column and / or membrane adsorber can be loaded with unpurified product while at least one other chromatography column and / or membrane adsorber can be regenerated or eluted, thereby providing a continuous and effective procedure of the method can be achieved.
In einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens erfolgen zwischen den wenigstens zwei Chromatographieschritten in (c) und/oder nach der Vireninaktivierung in Schritt (d) ein oder mehrere weitere Schritte zur Einstellung des pH-Wertes und/oder zur Einstellung der Leitfähigkeit und/oder Filtrations- und/oder Konzentrationsschritte und/oder ein Pufferaustausch. Dadurch wird eine an die Bedingungen anpassbare Fahrweise des Prozesses ermöglicht.In a further embodiment of the method according to the invention, one or more further steps for adjusting the pH and / or for adjusting the conductivity and / or filtration take place between the at least two chromatographic steps in (c) and / or after the virus inactivation in step (d) - and / or concentration steps and / or a buffer exchange. This allows a process-customizable driving style of the process.
Die mindestens eine Virenabreicherung des Schrittes d) kann insbesondere durch Einstellen des pH-Werts des partikel-freien Fluides erfolgen, bevorzugt auf einen pH-Wert von ≤ 4,0. Einstellen des pH-Wertes des zu inaktivierenden partikel-freien Fluids auf ≤ 4,0 kann beispielsweise durch Zugabe von HCl-Lösung erfolgen. Die Zugabe erfolgt typischerweise im Vorfeld der Vorrichtung zur Virusabreicherung. Üblicherweise wird der pH-Wert auf >4 mit einer Base beispielsweise Natriumhydroxid-Lösung (NaOH) eingestellt, um die Virusabreicherung zu beenden.The at least one virus removal of step d) can be carried out in particular by adjusting the pH of the particle-free fluid, preferably to a pH of ≦ 4.0. Setting the pH of the particle-free fluid to be inactivated to ≤ 4.0 can be carried out, for example, by adding HCl solution. The addition is typically in advance of the virus depletion device. Typically, the pH is adjusted to> 4 with a base, for example, sodium hydroxide solution (NaOH) to terminate the virus depletion.
Die mindestens eine Virenabreicherung des Schrittes d) kann aber auch durch eine solventdetergent Schritt erfolgen, bei dem eine Virenabreicherung durch Lösungsmittel- / Detergenz erzielt wird.The at least one virus depletion of step d) can also be carried out by a solvent detergent step, in which a virus depletion by solvent / detergent is achieved.
In einer weiteren Ausführungsform kann die Virenabreicherung auch durch UV-Behandlung und/oder durch thermische Behandlung erzielt werden.
Die mindestens eine Virenabreicherung des Schrittes d) kann insbesondere in einer Verweilstrecke erfolgen, in die ein segmentierter Produktstrom eingebracht werden kann.In a further embodiment, the virus depletion can also be achieved by UV treatment and / or by thermal treatment.
The at least one virus removal of step d) can be carried out in particular in a residence zone into which a segmented product stream can be introduced.
In einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens sind alle in den Schritten (a) bis (e) eingesetzten, produkt-berührten Elemente durch eine geeignete Keimreduktionsmethode keimreduziert.
Bevorzugt kann die Keimreduktionsmethode ausgewählt sein aus der Gruppe bestehend aus Gamma-Bestrahlung, Beta-Bestrahlung, Autoklavieren, Ethylenoxid (ETO)-Behandlung, Ozon-Behandlung (O3), Wasserstoffperoxid-Behandlung (H2O2) und Steam-in-place (SIP) Behandlung.In a further embodiment of the process according to the invention, all product-contacted elements used in steps (a) to (e) are germ-reduced by a suitable germ reduction method.
The germ reduction method may preferably be selected from the group consisting of gamma irradiation, beta irradiation, autoclaving, ethylene oxide (ETO) treatment, ozone treatment (O 3 ), hydrogen peroxide treatment (H 2 O 2 ) and steam-in-solution. place (SIP) treatment.
Entsprechend sind bevorzugt auch die in dem erfindungsgemäßen Verfahren eingesetzten Gegenstände und Elemente der Module, die mit Produktstrom in Kontakt treten, keimreduzierbar und/oder sterilisierbar, bevorzugt autoklavierbar, gamma-bestrahlbar, mit Ethylenoxid (ETO) begasbar, mit Ozon (O3) behandelbar, mit Wasserstoffperoxid (H2O2) behandelbar oder mit einer Steam-in-place (SIP)-Behandlung behandelbar, was einen keimreduzierten oder sogar aseptischen Betrieb des erfindungsgemäßen Verfahrens ermöglicht.Accordingly, the articles and elements of the modules used in the process according to the invention which come into contact with the product stream are preferably germ-reducible and / or sterilizable, preferably autoclavable, gamma-irradiated, gassed with ethylene oxide (ETO) and treatable with ozone (O 3 ) , treatable with hydrogen peroxide (H 2 O 2 ) or treatable with a steam-in-place (SIP) treatment, allowing germ-reduced or even aseptic operation of the method of the invention.
In einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens sind alle ab Filtrationsschritt (b) verwendeten produktberührten Elemente Einwegartikel oder werden als Einwegartikel benutzt. Solche eingesetzten Einwegartikel können dann im erfindungsgemäßen Verfahren auch als "disposabel" bzw. "Single-Use"-Artikel bezeichnet werden ("SU Technologie"). Dadurch wird der keimreduzierte Zustand des Verfahrens verbessert.In a further embodiment of the process according to the invention, all product-contacting elements used starting from filtration step (b) are disposable articles or are used as disposable articles. Such used disposable articles can then also be referred to as "disposable" or "single-use" articles in the process according to the invention ("SU technology"). This improves the germ-reduced state of the process.
In einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens werden alle Eingangsfluide durch einen Keimreduktionsfilter filtriert, wie etwa einen Sartoguard NF Filter der Firma Sartorius.In a further embodiment of the method according to the invention, all input fluids are filtered through a germ reduction filter, such as a Sartorius Sartoguard NF filter.
Dabei können bevorzugt alle Ausgänge durch eine Keimbarriere geschützt sein, die ein Rückwachstum von Mikroorganismen verhindern. Beispielsweise kann auch hier ein Sartoguard NF Filter der Firma Sartorius als Keimbarriere verwendet werden. Eine zusätzliche Sicherheit der Keimbarriere 11 kann durch eine Wechselschaltung von Filtern und/oder Abfallleitung erzielt werden. Eine weitere Maßnahme zur Gewährleistung der keimreduzierten Bedingungen kann durch eine periodische Sanitisierung der Abfallleitung vorzugsweise nach der Filtration mit z.B. NaOH Lösung erreicht werden. Weitere Verfahren wären UV Bestrahlung und Hitzebehandlung.In this case, all exits may preferably be protected by a germ barrier which prevents a re-growth of microorganisms. For example, a Sartorius NF filter from Sartorius can also be used as a germ barrier here. An additional security of the
Die modularen Verfahrensschritte des erfindungsgemäßen Verfahrens werden in einer weiteren Ausführungsform bevorzugt in Modulen durchgeführt, wobei die Module miteinander verbunden sind.
Dabei können bevorzugt die Module durch Verschweißen oder durch aseptische Konnektoren miteinander verbunden sein. Zum Verschweißen der Module kann z.B. das Gerät "TC Welder" der Firma Sartorius verwendet werden.The modular method steps of the method according to the invention are preferably carried out in modules in a further embodiment, wherein the modules are interconnected.
In this case, the modules may preferably be connected to one another by welding or by aseptic connectors. For welding the modules eg the device "TC Welder" of the company Sartorius can be used.
In einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens sind in den Schritten (a) bis (e) alle verwendeten Flüssigkeiten, Gase und Feststoffe keimreduziert. Dabei erfolgt die Keimreduktion bevorzugt durch eine Filtration durch einen Filter mit einer Porengröße von bevorzugt ≤ 0,45 µm. Dabei wird bevorzugt während des Verfahrens keine Inprocess-Sterilisierung durchgeführt. In anderen Ausführungsformen kann auch die Keimreduktion durch eine Filtration durch einen Filter mit einer Porengröße von bevorzugt ≤ 0,20 µm durchgeführt werden.In a further embodiment of the process according to the invention, all the liquids, gases and solids used are germ-reduced in steps (a) to (e). The germ reduction preferably takes place by filtration through a filter having a pore size of preferably ≦ 0.45 μm. In this case, preferably no in-process sterilization is carried out during the process. In other embodiments, the seed reduction may also be carried out by filtration through a filter having a pore size of preferably ≦ 0.20 μm.
In einer weiteren bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens wird vor dem Chromatographie-Schritt (c) ein Entgasen aller Fluide durchgeführt, welche auf die wenigstens zwei Chromatographiesäulen kommen, wobei das Entgasen vorzugsweise durch mindestens eine Bubble-Trap und/oder durch mindestens eine hydrophobe Mikrofiltrationsmembran über Vakuum und/oder durch Behandeln mit Ultraschall und/oder durch Durchperlen mit einem schlecht löslichen Gas, wie z.B. Helium erfolgt.In a further preferred embodiment of the method according to the invention, a degassing of all fluids is carried out before the chromatography step (c), which come to the at least two chromatography columns, wherein the degassing preferably by at least one bubble trap and / or by at least one hydrophobic microfiltration membrane Vacuum and / or by treatment with ultrasound and / or by bubbling with a poorly soluble gas, such as Helium takes place.
Dabei ist die Nutzung einer hydrophoben Mikrofiltrationsmembran über Vakuum für die Einhaltung der Keimfreiheit in der kontinuierlichen Führung des erfindungsgemäßen Verfahrens und der erfindungsgemäßen Anlage bevorzugt, da diese sich als besonders vorteilhaft im Vergleich zur Bubble-Trap erwiesen hat. Die verwendete hydrophobe Mikrofiltrationsmembran kann insbesondere ein MicroModule der Firma Membrana sein.The use of a hydrophobic microfiltration membrane via vacuum for maintaining the sterility in the continuous leadership of the invention Process and the system according to the invention preferred, since this has proven to be particularly advantageous compared to the bubble trap. The hydrophobic microfiltration membrane used can in particular be a MicroModule from Membrana.
In einer besonders bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens wird das partikel-freie Fluid aus Schritt a) mindestens einer Ultrafiltration gegen einen mikrobizid-haltigen Puffer unterzogen. Durch die Ultrafiltration werden die in dem Fluid enthaltenden Nährstoffe gegen einen mikrobizid-haltigen Puffer ausgetauscht, wodurch Mikroorganismen / Keimen die Wachstumsgrundlage in dem Fluid entzogen werden soll. Dadurch wird die Keimreduktion des Verfahrens zusätzlich verbessert.In a particularly preferred embodiment of the method according to the invention, the particle-free fluid from step a) is subjected to at least one ultrafiltration against a microbicide-containing buffer. By ultrafiltration, the nutrients contained in the fluid are exchanged for a microbicide-containing buffer, which microorganisms / germs should be removed from the growth base in the fluid. This further improves the germ reduction of the process.
Das dabei verwendete Mikrobizid, oder ein oder mehrere Mikrobizide können bevorzugt ausgewählt sein aus der Gruppe bestehend aus Imidazol, Benzoesäure, Sorbinsäure, para-Hydroxybenzoesäure-Ester, Sulfite, Disulfite, Azide, Orthophenylphenol, Nisin, Natamycin, Hexamethylentetramin, Dimethyldicarbonat, Nitrite, Nitrate, Essigsäure, Ascorbinsäure, Isoascorbinsäure, L-Milchsäure, Propionsäure, Borsäure und Lysozym.The microbicide used here, or one or more microbicides may preferably be selected from the group consisting of imidazole, benzoic acid, sorbic acid, para-hydroxybenzoic acid esters, sulfites, disulfites, azides, orthophenylphenol, nisin, natamycin, hexamethylenetetramine, dimethyldicarbonate, nitrites, nitrates , Acetic acid, ascorbic acid, isoascorbic acid, L-lactic acid, propionic acid, boric acid and lysozyme.
Die in dem mikrobizid-haltigen Puffer enthalten Mikrobizide können weiterhin sein ein oder mehrere Mikrobizide aus der Gruppe bestehend aus:
- E210 bis E213 Benzoesäure und ihre Salze, 0,05-0,1% in Lösungsmittel in saurem Milieu,
- 2-3g/kg in LM;
- E200 bis E203: Sorbinsäure und ihre Salze, 300-2000 mg/kg;
- E214 bis E219: PHB Ester (para-Hydroxybenzoesäure-Ester, Parabene), Butyl- und Propylparaben
- E220 bis E228: Sulfite und Disulfite,
- E231 und E232: Orthophenylphenol, 12 mg/kg;
- E234: Nisin,
- E235: Natamycin,
- E239: Hexamethylentetramin, 25mg/kg;
- E242: Dimethyldicarbonat;
- E249-E252: Nitrite und Nitrate, 300 mg/kg;
- E260: Essigsäure, 0,5-3%;
- E300-E302: Ascorbinsäure, 300 mg/kg;
- E315-E316: Isoascorbinsäure, 1500 mg/kg;
- E261-E263: Azetat;
- E270 L-Milchsäure;
- E280 bis E283: Propionsäure und ihre Salze, 1-3 g/kg;
- E284 und E285: Borsäure, max. 4g/kg;
- E1105 Lysozym; und
- Azide.
- E210 to E213 benzoic acid and its salts, 0.05-0.1% in solvent in acidic medium,
- 2-3g / kg in LM;
- E200 to E203: sorbic acid and its salts, 300-2000 mg / kg;
- E214 to E219: PHB esters (para-hydroxybenzoic acid esters, parabens), butyl and propyl paraben
- E220 to E228: sulfites and disulfites,
- E231 and E232: orthophenylphenol, 12 mg / kg;
- E234: Nisin,
- E235: natamycin,
- E239: hexamethylenetetramine, 25 mg / kg;
- E242: dimethyl dicarbonate;
- E249-E252: nitrites and nitrates, 300 mg / kg;
- E260: acetic acid, 0.5-3%;
- E300-E302: ascorbic acid, 300 mg / kg;
- E315-E316: isoascorbic acid, 1500 mg / kg;
- E261-E263: acetate;
- E270 L-lactic acid;
- E280 to E283: propionic acid and its salts, 1-3 g / kg;
- E284 and E285: boric acid, max. 4g / kg;
- E1105 lysozyme; and
- Azides.
In einer bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens ist das biopharmazeutische, biologischen makromolekulare Produkt ein Protein oder Peptid, dass ausgewählt ist aus der Gruppe bestehend aus monoklonalen Antikörpern, polyklonalen Antikörpern, rekombinanten Proteinen und Impfstoffen, bevorzugt DNA- und RNA-Impfstoffen.In a preferred embodiment of the method according to the invention, the biopharmaceutical, biological macromolecular product is a protein or peptide selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant proteins and vaccines, preferably DNA and RNA vaccines.
Die in Schritt c) verwendeten die Chromatographiesäulen und/oder Membranadsorber können jedes geeignete Bindungsprinzip aufweisen, etwa Affiniät des Produktes zu einem Liganden, ionische Wechselwirkungen, Metalchelat-Bindung, hydrophobe Interaktionen oder reine van-der-Waals-Kräfte. Beispielsweise kann der erster Chromatographieschritt der wenigstens zwei Chromatographieschritte eine Affinitätschromatographie sein (z.B. ein Ligand mit Affinität zum Produkt, wie z.B. Protein A, Protein G, Protein L, IgM, IgG und ein von Protein A, Protein G und Protein L, IgM, IgG unterschiedlichen rekombinanten Protein, welches eine Affinität für das Produkt hat). Danach folgt dann ein weiterer (zweiter) Chromatographieschritt, etwa eine Chromatographie über ionische Wechselwirkungen.
Das erfindungsgemäße Verfahren ist hier flexibel und kann in Schritt c) je nach zu erzielendem Reinheitsgrad und Konzentration des Produktes jedes geeignete Chromatographieprinzip in jeder Reihenfolge umfassen.
In einer besonders bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens umfassen die wenigstens zwei Chromatographiesäulen und/oder Membranadsorber des Schrittes (c) einen Ligand, der bevorzugt ausgewählt ist aus der Gruppe bestehend aus Protein A, Protein G, Protein L, IgM, IgG und einem von Protein A, Protein G und Protein L, IgM, und IgG unterschiedlichen rekombinanten Protein, welches eine Affinität für das Produkt hat.The chromatography columns and / or membrane adsorbers used in step c) may have any suitable binding principle, such as affinity of the product for a ligand, ionic interactions, metal chelate bonding, hydrophobic interactions or pure van der Waals forces. For example, the first chromatographic step of the at least two chromatography steps may be affinity chromatography (eg, a ligand having affinity for the product, such as protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, IgG different recombinant protein which has an affinity for the product). This is followed by another (second) chromatography step, such as chromatography on ionic interactions.
The process according to the invention is flexible here and can comprise in step c), depending on the degree of purity and concentration of the product to be achieved, any suitable chromatography principle in any order.
In a particularly preferred embodiment of the method according to the invention, the at least two chromatography columns and / or membrane adsorbers of step (c) comprise a ligand which is preferably selected from the group consisting of protein A, protein G, protein L, IgM, IgG and one of protein A, protein G and protein L, IgM, and IgG different recombinant protein, which has an affinity for the product.
In einer bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens weist das Verfahren der Schritte (a) bis (e) eine Laufzeit von mindestens 4 Stunden auf, bevorzugt von mindestens 8 Stunden, bevorzugt von mindestens 12 Stunden, bevorzugt von mindestens 24 Stunden, weiterhin bevorzugt von mindestens 48 Stunden, weiterhin bevorzugt von mindestens 7 Tagen, weiterhin bevorzugt von mindestens 4 Wochen, und besonders bevorzugt von mindestens 8 Wochen auf. Eine derart lange Laufzeit von mehreren Wochen kontinuierlicher Fahrweise wird nur ermöglicht durch die geschlossene, modulare und vor allem keimreduzierte Fahrweise des Prozesses.In a preferred embodiment of the process according to the invention, the process of steps (a) to (e) has a runtime of at least 4 hours, preferably of at least 8 hours, preferably of at least 12 hours, preferably of at least 24 hours, more preferably of at least 48 Hours, more preferably at least 7 days, more preferably at least 4 weeks, and most preferably at least 8 weeks. Such a long running time of several weeks of continuous driving is only made possible by the closed, modular and, above all, germ-reduced driving style of the process.
In einer bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens wird zwischen den Schritten a) bis e) und/oder danach mindestens ein Filtrationsschritt umfassend mindestens einen Filter durchgeführt.In a preferred embodiment of the method according to the invention, at least one filtration step comprising at least one filter is carried out between steps a) to e) and / or thereafter.
In einer besonders bevorzugten Ausführungsform des erfindungsgemäßen Verfahrens wird der Filter automatisch unter keimreduzierten Bedingungen gewechselt, wobei bevorzugt der automatische Filterwechsel die folgenden Schritte umfasst:
- (i) Umschalten des Flussweges auf einen neuen Filter bei Überschreiten eines Schwellenwertes am Drucksensor auf der Unfiltratseite unter Verschließen des Flussweges, wobei das Produkt im verbrauchten Filter bevorzugt durch ein Gas oder eine Flüssigkeit in die Filtratseite gedrückt wird,
oder bei Überschreiten einer maximalen Zeit des verbrauchten Filters im Flussweg, oder bei Überschreiten eines maximalen Volumens an Filtrat durch den verbrauchten Filter, - (ii) Entlüften des neuen Filters über eine Luftfilter mit einer Porengröße von bevorzugt <= 0,25 µm am Entlüftungsventil des neuen Filters, bevorzugt unter Beförderung von Produkt in den neuen Filter durch eine Feedpumpe, oder in einen keimreduziert angeschlossenen, geschlossenen Bag,
- (iii) Detektion der Beendigung der Entlüftung des neuen Filters auf der Unfiltratseite durch den Drucksensor oder einen Füllstandssensor oder eine Waage oder einen Flüssigkeitsdetektor,
- (iv) Öffnen des Filtratausgangs und Verschluss des Flussweges zwischen Entlüftungsventil und Luftfilter durch ein Ventil, und
- (v) Austausch des alten Filters gegen einen neuen Filter.
- (i) switching the flow path to a new filter when a threshold value is exceeded at the pressure sensor on the non-filtrate side, closing the flow path, wherein the product in the spent filter is preferably forced into the filtrate side by a gas or a liquid,
or if a maximum time of the spent filter in the flow path is exceeded, or if a maximum volume of filtrate through the spent filter is exceeded, - (ii) venting the new filter via an air filter having a pore size of preferably <= 0.25 μm at the venting valve of the new filter, preferably with transport of product into the new filter by a feed pump, or into a germ-reduced connected closed bag,
- (iii) detecting the completion of the venting of the new filter on the non-filtrate side by the pressure sensor or a level sensor or a balance or a liquid detector,
- (iv) opening the filtrate outlet and closing the flow path between the venting valve and air filter through a valve, and
- (v) Replacing the old filter with a new filter.
Die gleichzeitige oder nachgeschaltete Förderung von Produkt in den neuen Filter kann z.B. durch eine Feedpumpe erfolgen.The simultaneous or downstream delivery of product into the new filter may e.g. done by a feed pump.
Das bedeutet, dass in einer weiteren Ausführungsform des erfindungsgemäßen Verfahrens, der Filterwechsel (Umschalten von einem verbrauchten auf ein neues Filterelement) automatisch erfolgen kann. Als problematisch erweist sich hierbei die Filterentlüftung, die aber erforderlich ist, und die bei den zur Zeit erhältlichen Filtern manuell durchgeführt werden muss. Zunächst wird der Filter einer Keimreduktionsmethode durch ggf. ETO. Autoklavieren oder Gammabestrahlung unterworfen und dann mit dem Prozess verbunden. Danach kann der Filter auf der Unfiltratseite befüllt werden, während das Entlüftungsventil offen ist, welches nach dem erfolgreichen Entlüften geschlossen werden muss, damit die eigentliche Filtration durchgeführt werden kann. In einem Batch-Prozess ist eine stark keimreduzierte Handhabung auf der Unfiltratseite nicht erforderlich, da es in den kurzen Zeiträumen nur auf möglichst keimfreies Filtrat ankommt. In einem kontinuierlichen Prozess ist jedoch auch die möglichst keimfreie Führung der Unfiltratseite erforderlich, um eine Verkeimung des Prozesses zu verhindern. Im Stand der Technik muss das Entlüftungsventil durch eine Drehbewegung nach dem Befüllen des Filters manuell geschlossen werden, damit die eigentliche Filtration durchgeführt werden kann. Diese Drehbewegung ist nur sehr aufwendig zu automatisieren, da dabei auch noch eine axiale Verschiebung des Drehkörpers notwendig ist. Durch das axiale Verschieben des Drehkörpers samt der darauf angebrachten Dichtungen wird die Grenze verschoben. In Kombination mit einer vorher durchgeführten Keimreduktion ergibt sich das Problem, dass beim Keimreduzieren mit geschlossenem Entlüftungsventil die Keimgrenze bei Öffnen des Entlüftungsventils verschoben wird. Dabei wird ein keimbehafteter Bereich in den keimreduzierten Bereich eingebracht, und die Keimreduktion damit aufgehoben. Ein Keimreduzieren mit geöffnetem Filterventil ist nicht zu empfehlen, da die offene Position keine feste Position hat, und es dadurch leicht zu einer Beschädigung des Ventils kommen kann. Außerdem ist die offene Position meist wackelig, so dass es durch das normale Handling des Filters zu einer Verschiebung der Keimgrenze kommen kann.This means that in a further embodiment of the method according to the invention, the filter change (switching from a used to a new filter element) can take place automatically. The filter venting proves to be problematic in this case, but it is necessary and must be carried out manually in the filters currently available. First, the filter of a germ reduction method by possibly ETO. Subjected to autoclaving or gamma irradiation and then connected to the process. Thereafter, the filter can be filled on the unfiltrate side, while the vent valve is open, which must be closed after successful venting, so that the actual filtration can be performed. In a batch process, a strong germ-reduced handling on the unfiltrate side is not required because it arrives in the short periods only germ-free as possible filtrate. In a continuous process, however, the germ-free as possible management of Unfiltratseite is required to prevent bacterial contamination of the process. In the prior art, the vent valve must be manually closed by a rotary motion after filling the filter, so that the actual filtration can be performed. This rotational movement is very expensive to automate, since it also an axial displacement of the rotary body is necessary. The axial displacement of the rotary body together with the seals mounted thereon shifts the limit. In combination with a previously carried out germ reduction results in the problem that is displaced during the germ reduction with closed vent valve, the microbial limit when opening the vent valve. In this case, a germ-afflicted area is introduced into the germ-reduced area, and the germ reduction is thereby eliminated. Germinating with the filter valve open is not recommended because the open position has no fixed position, which can easily damage the valve. In addition, the open position is usually shaky, so that the normal handling of the filter can lead to a shift in the microbial limit.
Für die automatische Filterentlüftung ist es vorteilhaft, die Drehbewegung des Entlüftungsventils während der Inbetriebnahme zu vermieden, damit die Entlüftung nur durch einfache Maßnahmen durchgeführt werden kann. Das Entlüftungsventil kann nun so modifiziert werden, dass es auch im geschlossenen Zustand durchgängig ist, jedoch weiterhin zuverlässig zur Umwelt hin abdichtet. Das Ventil befindet sich dadurch im sicheren Zustand "geschlossen", was durch einen festen Sitz charakterisiert ist. Der Zustand "offen" ist meist nicht eindeutig definiert, da der Ventilkörper in der Führung stark wackelt und ein Verschieben der Grenze ermöglicht. An der Tülle des Drehkörpers wird ein Schlauch angebracht, der in einem hydrophoben <=0,2 µm Luftfilter endet. Diese Anordnung wird nun vorzugsweise durch ETO, Gammabestrahlung, Autoklavieren, oder Ozon (O3)-Behandlung oder durch Wasserstoffperoxid (H2O2)-Behandlung keimreduziert. Somit ist die gesamte Unfiltratseite bis zum Luftfilter am Entlüftungsventil keimreduziert bzw. keimarm. Zwischen Entlüftungsventil und Luftfilter wird der Schlauch in ein Schlauchquetschventil eingelegt, welches den Schlauch zuverlässig zu quetschen vermag. Die Entlüftung kann so vollautomatisch und keimarmst erfolgen. Der Filter wird befüllt, bis Flüssigkeit durch das Entlüftungsventil und den Schlauch in den Luftfilter eintritt. Der hydrophobe Entlüftungsfilter blockiert die Flüssigkeit. Gleichzeitig blockiert die Prozessanlage durch ein Ventil auf der Filtratseite den Produktionsstrom, so dass es zu einem Druckanstieg vor dem Filter kommt. Dieser wird über einen geeigneten Sensor detektiert. Ist ein bestimmter Schwellenwert des Drucks überschritten, wird das Quetschventil des Entlüftungsschlauches geschlossen, und das Ventil auf der Filtratseite geöffnet. In dieser Vorgehensweise kann mit einem modifizierten Entlüftungsventil der Filter automatisch ohne manuellen Eingriff modifiziert werden.
Dieser automatische Filterwechsel ist in
This automatic filter change is in
Das fertig gereinigte biopharmazeutischen, biologischen makromolekularen Produkte aus der heterogenen Zellkultur-Fluid-Mischung kann am Schluss des Verfahrens durch eine finale Filtration, bevorzugt durch einen Filter mit einer Porengröße von 0,2µm ein letztes Mal gefiltert werden. Die finale Filtration kann beispielsweise über gammabestrahlte Sartopore 2 Kapsulen (Midicap size 7, 0,05m2) in ein gammabestrahltes 5L GE ReadCircuit Bag erfolgen. Wenn der Füllstand des finalen Bags ausreichend ist, dann kann dieses Bag abgeschweißt werden und ein neues Bag an den Prozess angeschweißt werden.The final purified biopharmaceutical biological macromolecular products from the heterogeneous cell culture-fluid mixture can be filtered one final time at the end of the process by a final filtration, preferably through a filter having a pore size of 0.2 μm. The final filtration can be gamma-irradiated, for
Die Erfindung löst diese Aufgabe weiterhin durch Bereitstellen einer modularen Anlage zur kontinuierlichen, keimreduzierten Produktion und/oder Aufbereitung eines biopharmazeutischen, biologischen makromolekularen Produktes aus einer heterogenen Zellkultur-Fluid-Mischung, umfassend folgende Module:
- (a) mindestens ein Filtrationsmodul,
- (b) mindestens ein Chromatographiemodul, umfassend mindestens jeweils zwei Chromatographiesäulen und/oder Membranadsorbern,
- (c) mindestens ein Ultrafiltrationsmodul und/oder mindestens ein Diafiltrationsmodul und/oder mindestens ein Dialysemodul, und
- (d) mindestens ein Modul zur kontinuierlichen Virenabreicherung,
dadurch gekennzeichnet, dass die modulare Anlage geschlossen und keimreduziert ist.
- (a) at least one filtration module,
- (b) at least one chromatography module comprising at least two chromatography columns and / or membrane adsorbers,
- (c) at least one ultrafiltration module and / or at least one diafiltration module and / or at least one dialysis module, and
- (d) at least one module for continuous virus depletion,
characterized in that the modular system is closed and germ-reduced.
Das mindestens eine Chromatographiemodul, kann aber auch mehr als zwei Chromatographiesäulen und/oder Membranadsorbern umfassen, z.B. drei oder vier Chromatographiesäulen und/oder Membranadsorber.The at least one chromatography module, but may also comprise more than two chromatography columns and / or membrane adsorbers, e.g. three or four chromatography columns and / or membrane adsorbers.
In einer weiteren Ausführungsform der erfindungsgemäßen modularen Anlage sind alle in den Modulen (a) bis (d) eingesetzten und mit produktberührten Elemente durch eine Keimreduktionsmethode keimreduziert, wobei die Keimreduktionsmethode bevorzugt ausgewählt ist aus der Gruppe bestehend aus Gamma-Bestrahlung, Beta-Bestrahlung, Autoklavieren, Ethylenoxid (ETO)-Behandlung, Ozon-Behandlung (O3), Wasserstoffperoxid-Behandlung (H2O2) und steam-in-place (SIP) Behandlung.In a further embodiment of the modular system according to the invention, all of the modules (a) to (d) used and germinated with product contact elements by a germ reduction method, wherein the germ reduction method is preferably selected from the group consisting of gamma irradiation, beta irradiation, autoclaving , Ethylene oxide (ETO) treatment, ozone treatment (O 3 ), hydrogen peroxide treatment (H 2 O 2 ) and steam-in-place (SIP) treatment.
Bevorzugt sind die modular Anlage selber, sowie die Elemente der Module, die mit Produktstrom in Kontakt treten, keimreduzierbar und/oder sterilisierbar, bevorzugt autoklavierbar, gamma-bestrahlbar, mit Ethylenoxid (ETO) begasbar, mit Ozon (O3) behandelbar, mit Wasserstoffperoxid (H2O2) behandelbar oder mit einer Steam-in-place (SIP)-Behandlung behandelbar, was einen keimarmen oder sogar aseptischen Betrieb der erfindungsgemäßen modularen Anlage ermöglicht.Preferably, the modular system itself, as well as the elements of the modules that come into contact with product flow, germ-reducible and / or sterilizable, preferably autoclavable, gamma-irradiated, gassed with ethylene oxide (ETO), with ozone (O 3 ) treatable with hydrogen peroxide (H 2 O 2 ) treatable or with a steam-in-place (SIP) treatment treatable, which allows a germ-free or even aseptic operation of the modular system according to the invention.
In einer weiteren Ausführungsform der erfindungsgemäßen modularen Anlage sind alle in den Modulen (a) bis (d) verwendeten produktberührten Gegenstände Einwegartikel oder werden als Einwegartikel benutzt. Dabei sind die Module bevorzugt durch Verschweißen oder durch aseptische Konnektoren miteinander verbunden. Beispielsweise sind aseptische "ReadyMate"-Konnektoren der Firma GE bevorzugt in der erfindungsgemäßen modularen Anlage verwendete aseptische Konnektoren.
Bevorzugt werden fertig einsetzbare Einwegartikel ("Disposables", "ready-to-use") als gammabestrahlte Elemente verwendet.In a further embodiment of the modular system according to the invention, all product-contacted articles used in the modules (a) to (d) are disposable articles or are used as disposable articles. The modules are preferably connected to one another by welding or by aseptic connectors. For example, aseptic "ReadyMate" connectors from GE are preferably aseptic connectors used in the modular system according to the invention.
Prefabricated disposable articles ("disposables", "ready-to-use") are preferably used as gamma-irradiated elements.
In einer bevorzugten Ausführungsform der erfindungsgemäßen modularen Anlage gehen alle Eingangsfluide durch einen Keimreduktionsfilter, wobei bevorzugt alle Ausgänge durch eine Keimbarriere geschützt sind, die ein Rückwachstum verhindern.In a preferred embodiment of the modular system according to the invention, all input fluids pass through a germ reduction filter, wherein preferably all exits are protected by a germ barrier which prevents regress growth.
Das fertig gereinigte biopharmazeutischen, biologischen makromolekularen Produkte aus der heterogenen Zellkultur-Fluid-Mischung kann am Ende der modularen Anlage durch eine finale Filtration, bevorzugt durch einen Filter mit einer Porengröße von 0,2µm ein letztes Mal gefiltert werden. Die finale Filtration kann beispielsweise über gammabestrahlte Sartopore 2 Kapsulen (Midicap size 7, 0,05m2) in ein gammabestrahltes 5L GE ReadCircuit Bag erfolgen. Wenn der Füllstand des finalen Bags ausreichend ist, dann kann dieses Bag abgeschweißt werden und ein neues Bag an den Prozess angeschweißt werden.The final purified biopharmaceutical biological macromolecular products from the heterogeneous cell culture fluid mixture can be filtered one final time at the end of the modular unit through a final filtration, preferably through a filter having a pore size of 0.2 μm. Final filtration can be done, for example, via gamma-
Die vorliegende Erfindung einschließlich bevorzugter Ausführungsformen wird in Verbindung mit den nachfolgenden Zeichnungen und dem Beispiel erläutert, ohne hierauf beschränkt zu sein. Die Ausführungsformen können beliebig miteinander kombiniert werden, sofern sich nicht eindeutig das Gegenteil aus dem Kontext ergibt.The present invention, including preferred embodiments thereof, is illustrated in conjunction with the following drawings and example, but is not limited thereto. The embodiments can be combined as desired, unless clearly the opposite results from the context.
Es zeigen:
-
FIG. 1 zeigt schematisch ein Verfahrensschema einer Ausführungsform des erfindungsgemäßen Verfahrens. Die Zahlen in Klammern sind Verweise auf die Massenbilanz, wie inBeispiel 1 und Tabelle 1 aufgeführt. -
Fig. 2 zeigt schematisch das Arbeitsprinzip des Filtrationsschrittes des Verfahrens und der Anlage unter Ersetzen eines verbrauchtenFilters 17 durch Einschweißen eines neuen Filters 16. -
Fig. 3 zeigt exemplarisch die zwei Verfahrensschritte Vireninaktivierung und Neutralisierung - zwei Units die modular aufgebaut sind, wobei die pH Sonden pH0501 und pH0502 autoklaviert werden und der Rest jeweils gammabestrahlt wird. Die pH Sonden pH0501 und pH0502 werden dann in ein Assembly eingeschweißt. Die Bags sind über aseptische GE ReadyMate® Konnektoren angeschlossen. Die Verbindung zu Prot-A und der Filtrationseinheit ist zunächst zugeschweißt und wird dann an die jeweiligen Einheiten angeschweißt. -
Fig. 4 zeigt exemplarisch den modularen Aufbau der Anlage, wobei alle Eingangsströme und Ausgangsströme über eine Keimbarriere 10, 13 mit der Umgebung verbunden sind. Die exemplarische modulare Anlage 1 besteht aus drei Filtrationsmodulen 2 mit jeweils zwei wechselseitig betriebenen Filtern 13,zwei Chromatographiemodule 3mit zwei Chromatographiesäulen 4 bzw.zwei Membranadsorber 5 ein Virenabreicherungschritt z.B.Vireninaktivierung 9,einem Ultrafiltrationsmodul 6 und einem Diafiltrationsmodul 7. Die Puffer werden über einen hydrophobenFilter 15 oder Bubble-trap 14 von Gasblasen befreit.
-
FIG. 1 schematically shows a process scheme of an embodiment of the method according to the invention. The numbers in parentheses are references to the mass balance as listed in Example 1 and Table 1 . -
Fig. 2 schematically shows the principle of operation of the filtration step of the method and the system with replacement of a spentfilter 17 by welding anew filter 16. -
Fig. 3 shows an example of the two process steps virus inactivation and neutralization - two units that are modular, with the pH probes pH0501 and pH0502 are autoclaved and the rest is gamma irradiated each. The pH probes pH0501 and pH0502 are then welded into an assembly. The bags are connected via aseptic GE ReadyMate ® connectors. The connection to Prot-A and the filtration unit is first welded shut and then welded to the respective units. -
Fig. 4 shows an example of the modular design of the system, wherein all input currents and output currents are connected via agerm barrier modular plant 1 consists of threefiltration modules 2 with two mutually operated filters 13, twochromatography modules 3 with twochromatography columns 4 or two membrane adsorbers 5 a virus depletion stepeg virus inactivation 9, anultrafiltration module 6 and adiafiltration module 7. The buffers arehydrophobic Filter 15 orbubble trap 14 freed of gas bubbles.
Tabelle 1 zeigt die gemittelten Flussraten und Antikörper-Konzentrationen der in
Die verwendeten Bezugsziffern sind:
- 1 = Modulare Anlage
- 2 = Filtrationsmodul
- 3 = Chromatographiemodul
- 4 = Chromatographiesäule
- 5 = Membranadsorber
- 6 = Ultrafiltrationsmodul
- 7 = Diafiltrationsmodul
- 8 = Dialysemodul
- 9 = Virenabreicherung
- 10 = Keimreduktionsfilter
- 11 = Keimbarriere
- 12 = aseptischer Konnektor
- 13 = Filter mit einer Porengröße von bevorzugt ≤ 0,45 µm
- 14 = Bubble-Trap
- 15= hydrophobe Mikrofiltrationsmembran
- 16 = neuer Filter
- 17 = verbrauchter Filter
- 18 = Drucksensor
- 19 = Luftfilter, Porengröße bevorzugt <= 0,25 µm
- 20 = Entlüftungsventil des neuen Filters 16,
- 21 = Feedpumpe
- 22 = Füllstandsensor
- 23 = Waage
- 24 = Ventil
- 25 = Abfallstrom
- 26 = Produktstrom
- 27 = Puffer
- 28 = Flüssigkeitsdetektor
- 29 = Bag
- 1 = modular system
- 2 = filtration module
- 3 = chromatography module
- 4 = chromatography column
- 5 = membrane adsorber
- 6 = ultrafiltration module
- 7 = Diafiltration module
- 8 = dialysis module
- 9 = virus depletion
- 10 = germ reduction filter
- 11 = germ barrier
- 12 = aseptic connector
- 13 = filter with a pore size of preferably ≤ 0.45 microns
- 14 = bubble trap
- 15 = hydrophobic microfiltration membrane
- 16 = new filter
- 17 = used filter
- 18 = pressure sensor
- 19 = air filter, pore size preferably <= 0.25 microns
- 20 = vent valve of the
new filter 16, - 21 = feed pump
- 22 = level sensor
- 23 = Libra
- 24 = valve
- 25 = waste stream
- 26 = product flow
- 27 = buffer
- 28 = liquid detector
- 29 = bag
Um ein Protein kontinuierlich und keimreduziert aus einer heterogenen Zellkultur-Fluid-Mischung zu reinigen, wurde eine Miniplant mit folgenden Modulen und zugehörigen Verfahrensschritten aufgebaut:
- Wenn nicht anders vermerkt wurden im Prozess MasterFlex-Schlauchpumpen mit einem EasyLoad-II Pumpenkopf verwendet. Als Schläuche wurden Masterflex LS16 bzw. Cflex oder Sanipure verwendet. Alle eingesetzten produktberührten Bauteile wurden mit 25kGy Gamma-bestrahlt. In Ausnahmen, wenn Gammabestrahlung materialtechnisch nicht zulässig war, wurden Komponenten bei 121°C für 20min autoklaviert. Teilassemblies mit pH-Sonden oder Virenfilter. Wenn möglich wurden fertig einsetzbare Einwegartikel ("Disposables", "ready-to-use") als gammabestrahlte Module verwendet. Dies war ausnahmslos bei allen Beuteln ("Bags") der Fall. Diese Bags wurden in der Regel mit ReadyMate® Konnektoren der Firma General Electric (GE) an die Module verbunden. Zwischen jedem Modul wurde eine Single-Use gammabestrahlte Bag (ReadyCircuit 1L, GE) als Ausgleichsbehälter zwischen dem Ausgangsstrom des Moduls n-1 und dem Eingangsstrom des Moduls n platziert. In der Regel existierte zu dem Zeitpunkt in jedem Modul ein Eingangs- und ein Ausgangsstrom. Wo eine Entlüftung der Produktflüssigkeit vorteilhaft war, wurden die Behälter über einen hydrophoben 0,2 µm Filter von der Umgebung abgeschlossen.
- Unless otherwise noted, the process used MasterFlex peristaltic pumps with an EasyLoad-II pumphead. As hoses Masterflex LS16 or Cflex or Sanipure were used. All product wetted components used were gamma-irradiated with 25kGy. In exceptional cases, when gamma irradiation was not technically feasible, components were autoclaved at 121 ° C for 20 minutes. Subassemblies with pH probes or virus filters. Wherever possible, disposable disposables ("disposables", "ready-to-use") have been used as gamma-irradiated modules. This was without exception the case with all bags. These bags were usually connected to the modules using ReadyMate ® connectors from General Electric (GE). Between each module, a single-use gamma-irradiated bag (ReadyCircuit 1L, GE) was placed as a surge tank between the output current of module n-1 and the input current of module n. As a rule, there was an input and an output current in each module at that time. Where venting of the product liquid was beneficial, the containers were sealed from the environment via a hydrophobic 0.2 μm filter.
Zur kontinuierlichen Herstellung eines monoklonalen Antikörpers IgG wurde ein 10 L Perfusionsreaktor verwendet. Die viable Zelldichte betrug im stationären Zustand 60-70 Mio Zellen / mL. Der Titer betrug ∼115 mg/L . Die Produktion wurde mit zwei parallelen Perfusionsreaktoren für 28 Tage betrieben.For the continuous production of a monoclonal antibody IgG, a 10 L perfusion reactor was used. The viable cell density in the stationary state was 60-70 million cells / mL. The titer was ~115 mg / L. The production was operated with two parallel perfusion reactors for 28 days.
Das Produkt wurde kontinuierlich über einen Schrägplattenabscheider (Settler) abgeführt, durch den ein Großteil der Zellen zurückgehalten wurde.The product was continuously removed via a slant plate separator (settler), which retained most of the cells.
Die Klärfiltration erfolgte über parallel betriebene Sartoguard NF 0,2µm Filter (T-style, MaxiCap, 0,65m2).
Das Filtrat aus der i) Zell-Klärfiltration wurde zunächst über eine Ultrafiltrations-Hohlfaser-Membran (GE Healthcare ReadytoProcess, 0,2m2, gammabestrahlt) kontinuierlich um den Faktor 10 aufkonzentriert. Als Kreislaufpumpe diente eine disposable QuattroFlow 1200 SU Pumpe, dessen Pumpenkopf vor der Gammabestrahlung in das Schlauchassembly eingebunden wurde.The filtrate from i) cell clarification filtration was first passed through an ultrafiltration hollow fiber membrane (GE Healthcare Ready to Process, 0.2m 2 , gamma-irradiated). continuously concentrated by a factor of 10. The circulation pump was a disposable QuattroFlow 1200 SU pump whose pump head was integrated into the hose assembly before the gamma radiation.
Die Medienbestandteile des konzentrierten Produkts wurde dann mit einem 50mM Imidazol/NaCl Puffer über eine Gambro Revaclear300 Dialysemembran ausgetauscht. Das Modul wird vom Hersteller steril verpackt geliefert und wurde unter einer Sterilbank mit dem gammabestrahlten Schlauchassembly verbunden. Das Permeat aus der Aufkonzentrierung und der medienbehaftete Abfallstrom wurden in einen gammabestrahlten 200L Sartorius Flexboy geleitet. Das Auswechseln des Flexboys wurde durch Umschweißen mit einem Sartorius Schweißgerät durchgeführt.The media components of the concentrated product were then exchanged with a 50 mM imidazole / NaCl buffer over a Gambro Revaclear 300 dialysis membrane. The module is supplied sterile packaged by the manufacturer and connected to the gamma irradiated hose assembly under a sterile bench. The permeate from the concentration and the media-laden waste stream were fed into a gamma-irradiated 200L Sartorius Flexboy. The replacement of the Flexboys was carried out by welding with a Sartorius welding machine.
Das Produkt wurde vor der Abfüllung kontinuierlich mit gammabestrahlten Sartoguard NF Filtern (MaxiCap Size 8) im Wechselbetrieb in ein 200L Flexboy filtriert. Aufbau und Betrieb war Analog zu B. Downstream DSP-1 i) Zell Klärfiltration.The product was continuously filtered with gamma-irradiated Sartoguard NF filters (MaxiCap Size 8) in a 200L Flexboy before filling. Construction and operation was analogous to B. Downstream DSP-1 i) cell clarification filtration.
Nach der Lagerung wurde das Produkt aus dem DSP-1 nochmals filtriert, um die nachgeschalteten Chromatographiesäulen vor Partikel zu schützen.After storage, the product from the DSP-1 was filtered again to protect the downstream chromatography columns from particles.
Mabselect Sure (GE) wurde als Prot-A Harz zum Isolieren des IgG benutzt. Dabei wurde der IgG bis zu Faktor 10 aufkonzentriert und der größte Teil der Kontaminanten entfernt. Eine kontinuierliche BioSMB-Anlage der Firma Tarpon Biosystems, Inc. wurde benutzt mit 12 Säulen (ID 16 mm, L 80 mm), wobei 8 Säulen in der Ladezone waren (2 Säulen in Serie und 4 parallele Serien). Der gesamte Flussweg inklusive der Säulen wurde durch Sanitisierung oder gamma-Bestrahlung keimarm gemacht. Die Beladung pro Zyklus betrug 32 Säulenvolumina pro Säule. Als Puffer wurden Acetat Puffer mit unterschiedlichen Molarität, pH und Leitfähigkeiten eingesetzt. Sämtliche Puffer wurden über einen gammabestrahlten oder autoklavierten 0,2µm Filter in ein gammabestrahltes Bag filtriert. Die Ausgangsschläuche der Pufferbags wurden an die Eingänge der BioSMB-Anlage geschweißt. Diese hatte an Ihren Eingängen jeweils eine gammabestrahlte Entgaser Membran (Liquicell Micro Module, Membrana). In gleicherweise wurde die Produktleitung über einen solchen Entgaser an den Eingang der BioSMB-Anlage angeschweißt. Über eine Vakuumpumpe mit 50mbar wurden dann alle Eingangsströme entgast.Mabselect Sure (GE) was used as Prot-A resin to isolate the IgG. The IgG was concentrated up to a factor of 10 and most of the contaminants were removed. A continuous BioSMB plant from Tarpon Biosystems, Inc. was used with 12 columns (
Das Virus-Inaktivierung und Neutralisierung bestand aus drei Modulen und befand sich zwischen der Capture Chromatography und einer 0,2µm Filtration: (a) eine Homogenisierungsschleife mit einer Schlauchpumpe M0502 (b) einer Verweilzeitschleife schematisch als gewickelter Schlauch dargestellt (c) einem Neutralisierungsbag in dem der pH auf 7,5 eingestellt werden konnte. Die Module wurden entsprechend der Schweißstellen in
Leitungsabschnitt mit pH Sonden, hier pH0501 und pH0502, wurden autoklaviert. pH Sonden Abschnitte wurden dann in Assemblies eingeschweißt.
Bags wurden wie gezeigt über aseptische GE ReadyMate® Konnektoren angeschlossen. Die Verbindungen zur Protein A Eluat-Leitung und Filtrationsmodul waren zunächst zugeschweißt und wurden dann an die jeweiligen Module angeschweißt.Line section with pH probes, here pH0501 and pH0502, were autoclaved. pH probe sections were then sealed in assemblies.
Bags were shown connected via aseptic GE Ready Mate ® connectors. The connections to the protein A eluate line and filtration module were initially sealed and then welded to the respective modules.
Proteine konnten nach einem pH-Shift präzipitieren, wobei das präzipitierte Protein abfiltriert wurde.Proteins were able to precipitate after a pH shift, whereby the precipitated protein was filtered off.
Das Produkt aus der obigen 0,2µm Filtration wurde über zwei Chromatographieschritte durch zunächst vier sequentiell (4-PCC) betriebenen 2,5ml Capto Adhere (2,5ml GE) und dann zwei wechselseitig betriebenen 20ml Anionentauscher (Pall Hypercel StarAX) aufgereinigt. Dabei wurden ProtA Leachables, DNA, HCP und Aggregate abgereinigt. Die zwei Chromatographieschritte wurden über ein gammabestrahles Bag (GE ReadyCircuit® 1 L) miteinander verbunden, in dem durch Zufuhr von Wasser die Leitfähigkeit entsprechend der Anforderung des Anionentauscher auf 7,5mS/cm eingestellt wurden. Der gesamte Flussweg inklusive der Säulen wurde sanitisiert oder gammabestrahlt. Die Beladung pro Zyklus betrug 50 Säulenvolumina pro Säule. Als Puffer wurden Acetat Puffer mit unterschiedlichen Molarität, pH und Leitfähigkeiten eingesetzt. Sämtliche Puffer wurden über einen gammabestrahlten oder autoklavierten 0,2µm Filter in ein gammabestrahltes Bag filtriert. Die Ausgangsschläuche der Pufferbags wurden an die Eingänge der BIO- geschweißt. Diese hatte an Ihren Eingängen jeweils eine gammabestrahlte Entgasungsmembran (Liquicell Micro Module, Membrana), In gleicherweise wurde die Produktleitung der obigen 0,2µm Filtration über einen solchen Entgaser an den Eingang der BioSMB-Anlage angeschweißt. Über eine Vakuumpumpe mit 50mbar wurden dann alle Eingangsströme entgast. Der Abfallstrom wurde in einen gammabestrahlten 200L Sartorius Flexboy geleitet. Das Auswechseln des Flexboys wurde durch Umschweißen mit einem Sartorius Schweißgerät durchgeführt.
Der Produktstrom wurde wiederum in einem gammabestrahlten Produktbag (GE ReadyCircuit 1 L) aufgefangen.The product from the above 0.2μm filtration was passed through two chromatographic steps by first using four sequentially (4-PCC) 2.5ml Capto Adhere (2.5ml GE) and then two 20ml alternating anion exchangers (Pall Hypercel StarAX). purified. ProtA leachables, DNA, HCP and aggregates were purified. The two chromatography steps were a gammabestrahles Bag (GE Circuit Ready ® 1 L) connected with each other, in which the conductivity were adjusted according to the requirement of the anion exchanger on 7,5mS / cm by adding water. The entire river route including the columns was sanitized or gamma-irradiated. The loading per cycle was 50 column volumes per column. As buffer acetate buffer with different molarity, pH and conductivities were used. All buffers were filtered through a gamma-irradiated or autoclaved 0.2μm filter into a gamma-irradiated bag. The outlet hoses of the buffer bags were welded to the inputs of the BIO. Each had a gamma-irradiated degassing membrane (Liquicell Micro Module, Membrana) at its inputs. In the same way, the product line of the above 0.2μm filtration was welded to the entrance of the BioSMB plant via such a degasser. All input streams were then degassed via a 50mbar vacuum pump. The waste stream was directed into a gamma-irradiated 200L Sartorius Flexboy. The replacement of the Flexboys was carried out by welding with a Sartorius welding machine.
The product stream was again collected in a gamma-irradiated product bag (GE ReadyCircuit 1 L).
Aus dem Produktbag der Polish Chromatographie wurde die Produktlösung zunächst über eine 0,1 µm Kapsule (Sartopore2, MidiCap size 9, 0,2m2) vorfiltriert. Durchführung und Aufbau waren analog zu der B. Downstream DSP-1 i) Zell Klärfiltration.The product solution was first prefiltered from the product bag of the Polish Chromatography via a 0.1 μm capsule (Sartopore2,
Die Ausgangsleitung aus der Vorfiltration wurde direkt über eine Schlauchpumpe mit dem Eingang der Virusfiltration aus C. Downstream DSP-II über Verschweißen verbunden. Ansonsten war der Aufbau und Betrieb der Virusfiltration aus C. Downstream DSP-II analog zu C. Downstream DSP-II 0,2µm Filtration. Jedoch wurde als Virusfilter ein Virosart CPV Filter (MidiCap size 9, 0,2m2) verwendet, der gemäß der Herstellemorschrift eingespült und autoklaviert wurde. Wiederum wurden die Filter in das Assembly eingeschweißt. Der Produktstrom wurde wiederum in ein gammabestrahles Produktbag (GE ReadyCircuit 1 L) gepumpt.The output line from the prefiltration was connected directly via a peristaltic pump to the input of virus filtration from C. Downstream DSP-II via welding. Otherwise, the setup and operation of virus filtration from C. Downstream DSP-II was analogous to C. Downstream DSP-II 0.2μm filtration. However, the virus filter used was a Virosart CPV filter (
Die finale Konzentration und Umpufferung war analog zu der obigen B. DSP-I ii) Konzentration und Umpufferung aufgebaut und unterschied sich nur dadurch, dass eine autoklavierte UV-Zelle zur Überwachung der Produktkonzentration in die Konzentrationsschleife eingebunden war. Die Umpufferung erfolgte ebenfalls analog zu der obigen B. DSP-I ii) Konzentration und Umpufferung, wobei hier ein 50mM Phosphatpuffer pH 7,5 eingesetzt wurden. Der Produktstrom wurde wiederum in ein gammabestrahles Produktbag (GE ReadyCircuit 1 L) gepumpt.The final concentration and rebuffering was analogous to the above B. DSP-I ii) concentration and rebuffering and differed only in that an autoclaved UV cell was involved in monitoring the product concentration in the concentration loop. The rebuffering was also carried out analogously to the above B. DSP-I ii) concentration and rebuffering, here a 50mM phosphate buffer pH 7.5 were used. The product stream was pumped again into a gamma-irradiated product bag (GE ReadyCircuit 1 L).
Die finale Filtration erfolgte wie oben in B. DSP-1 i) über gammabestrahlte Sartopore 2 Kapsulen (Midicap size 7, 0,05m2) in ein gammabestrahltes 5L GE ReadCircuit Bag. Wenn der Füllstand des finalen Bags ausreichend war, wurde dieses Bag abgeschweißt und ein neues Bag an den Prozess angeschweißt.Final filtration was as above in B. DSP-1 i) via gamma-
Eine regelmäßig gefahrene Laufzeit des erfindungsgemäßen Verfahrens, wie in Beispiel 1 beispielhaft beschrieben, war 3 Tage ohne Keimwachstum, wobei die Chromatographiesäulen durch eine 40 % Isopropanol + 0.5 M NaOH sanitisiert wurden. Bei Laufzeiten des erfindungsgemäßen Verfahrens von über 3 Tage wurden die Chromatographiesäulen gamma -bestrahlt.A regularly run time of the method according to the invention, as described by way of example in Example 1, was 3 days without germ growth, wherein the chromatography columns were sanitized by a 40% isopropanol + 0.5 M NaOH. At run times of the inventive method of more than 3 days, the chromatography columns were gamma-irradiated.
Die gemittelten Flussraten und Antikörperkonzentrationen der in 
Die Arbeiten, die zu dieser Anmeldung geführt haben, wurden gemäß der Finanzhilfevereinbarung "Bio.NRW: MoBiDiK - Modulare Bioproduktion - Disposable und Kontinuierlich" im Rahmen des Europäischen Fonds für regionale Entwicklung (EFRE) gefördert.The work leading to this notification has been funded under the grant agreement "Bio.NRW: MoBiDiK - Modular Bioproduction - Disposable and Continuous" under the European Regional Development Fund (ERDF).
Claims (18)
dass das Verfahren geschlossen und modular durchgeführt wird.A process for the continuous, germ-reduced production and / or processing of a biopharmaceutical, biological macromolecular product from a heterogeneous cell culture-fluid mixture, comprising the steps:
that the process is closed and modular.
Priority Applications (19)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP15166686.4A EP3015542A1 (en) | 2015-05-07 | 2015-05-07 | Modular system and method for continuous, germ reduced production and/or processing of a product |
| CA2984981A CA2984981A1 (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| AU2016257253A AU2016257253B2 (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| MX2017014278A MX2017014278A (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner. |
| JP2017557944A JP6794372B2 (en) | 2015-05-07 | 2016-04-29 | Modular systems and methods for continuously producing and / or preparing products in a sterilized state |
| SG11201709094WA SG11201709094WA (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| PCT/EP2016/059700 WO2016177650A1 (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| CN201680039860.3A CN107995924B (en) | 2015-05-07 | 2016-04-29 | Modular device and method for the continuous and microbial-reduced production and/or treatment of products |
| RU2017142617A RU2721535C2 (en) | 2015-05-07 | 2016-04-29 | Method for continuous microorganism-reducing production and/or treatment of product |
| KR1020177034919A KR20180002783A (en) | 2015-05-07 | 2016-04-29 | Modular systems and methods for continuous, germ-reducing production and / or processing of products |
| EP16722580.4A EP3292195A1 (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| BR112017023912-4A BR112017023912A2 (en) | 2015-05-07 | 2016-04-29 | modular system and process for continuous production and / or processing of a disinfected product |
| US15/571,754 US10696940B2 (en) | 2015-05-07 | 2016-04-29 | Modular system and process for the continuous, microbe-reduced production and/or processing of a product |
| ARP160101271A AR104523A1 (en) | 2015-05-07 | 2016-05-04 | PLANT AND MODULAR METHOD FOR THE PRODUCTION AND / OR CONTINUOUS TREATMENT OF STERILE PRODUCTS |
| TW105113911A TWI696696B (en) | 2015-05-07 | 2016-05-05 | Modular system and process for the continuous, microbe-reduced production and/or processing of a product |
| IL255383A IL255383B (en) | 2015-05-07 | 2017-11-01 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| SA517390299A SA517390299B1 (en) | 2015-05-07 | 2017-11-07 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| ZA2017/08284A ZA201708284B (en) | 2015-05-07 | 2017-12-06 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
| HK18114001.8A HK1254902A1 (en) | 2015-05-07 | 2018-11-02 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP15166686.4A EP3015542A1 (en) | 2015-05-07 | 2015-05-07 | Modular system and method for continuous, germ reduced production and/or processing of a product |
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| EP3015542A1 true EP3015542A1 (en) | 2016-05-04 |
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| EP15166686.4A Withdrawn EP3015542A1 (en) | 2015-05-07 | 2015-05-07 | Modular system and method for continuous, germ reduced production and/or processing of a product |
| EP16722580.4A Withdrawn EP3292195A1 (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
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| EP16722580.4A Withdrawn EP3292195A1 (en) | 2015-05-07 | 2016-04-29 | Modular system and method for continuously producing and/or preparing a product in a disinfected manner |
Country Status (18)
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| US (1) | US10696940B2 (en) |
| EP (2) | EP3015542A1 (en) |
| JP (1) | JP6794372B2 (en) |
| KR (1) | KR20180002783A (en) |
| CN (1) | CN107995924B (en) |
| AR (1) | AR104523A1 (en) |
| AU (1) | AU2016257253B2 (en) |
| BR (1) | BR112017023912A2 (en) |
| CA (1) | CA2984981A1 (en) |
| HK (1) | HK1254902A1 (en) |
| IL (1) | IL255383B (en) |
| MX (1) | MX2017014278A (en) |
| RU (1) | RU2721535C2 (en) |
| SA (1) | SA517390299B1 (en) |
| SG (1) | SG11201709094WA (en) |
| TW (1) | TWI696696B (en) |
| WO (1) | WO2016177650A1 (en) |
| ZA (1) | ZA201708284B (en) |
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| AR104523A1 (en) | 2017-07-26 |
| CN107995924B (en) | 2021-09-07 |
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| AU2016257253B2 (en) | 2020-06-18 |
| JP6794372B2 (en) | 2020-12-02 |
| KR20180002783A (en) | 2018-01-08 |
| CN107995924A (en) | 2018-05-04 |
| TW201706403A (en) | 2017-02-16 |
| US20180135006A1 (en) | 2018-05-17 |
| IL255383B (en) | 2021-09-30 |
| CA2984981A1 (en) | 2016-11-10 |
| MX2017014278A (en) | 2018-03-07 |
| US10696940B2 (en) | 2020-06-30 |
| RU2017142617A (en) | 2019-06-10 |
| WO2016177650A1 (en) | 2016-11-10 |
| BR112017023912A2 (en) | 2018-07-17 |
| IL255383A0 (en) | 2017-12-31 |
| SG11201709094WA (en) | 2017-12-28 |
| HK1254902A1 (en) | 2019-08-02 |
| SA517390299B1 (en) | 2021-03-02 |
| RU2721535C2 (en) | 2020-05-19 |
| JP2018518161A (en) | 2018-07-12 |
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